DNA REPLICATION AND REPAIR

Mr. Steven Sekulya

 DNA REPLICATION

The process of copying of base sequence present in the parent

strand to daughter strand, thereby passing the genetic
information from parent to progeny is called Replication
 Salient Features of DNA Replication
❑Occurs in the S Phase of the cell cycle
❑DNA strands separate and each acts as template strand on which
complementary strand is synthesized
❑Base pairing rule is obeyed
❑Semiconservative
‒ Half of the parent strand is conserved in the daughter DNA
❑New Strand is synthesized always in 5’ to 3’ direction
❑Overall DNA replication is bidirectional
❑Synthesis of DNA in both strands are not similar
‒ Leading Strand: The strand on which DNA is continuously polymerized
‒ Lagging Strand: The strand on which DNA is discontinuously polymerized.
 Proteins and Enzymes Involved in the DNA Replication
❑Topoisomerases
‒ Relieve torsional strain that results from helicase induced unwinding of DNA
‒ It’s a Nicking Resealing Enzyme
❑Helicase: ATP driven processive unwinding of DNA
DnaB is the principal helicase of replication in E. coli. Binding of this hexameric protein to
DNA requires DnaC
❑Single Strand Binding (SSB) protein prevents premature reannealing of dsDNA.
❑DnaA protein initiates replication by binding to specific nucleotide sequences (DnaA
boxes) within oriC causing an AT-rich region in the origin to melt
❑DNA Primase: Initiates synthesis of RNA primers Special class of DNA dependent RNA
Polymerase
❑ DNA Polymerase: Catalyse the chemical reaction of DNA Polymerization. Synthesizes DNA
only in 5’ to 3’ direction
❑ DNA Ligase: Seals the single strand nick between the nascent chain and Okazaki
fragments on lagging strand.
 DNA POLYMERASES

❑Enzymes that catalyse Deoxyribonucleotide polymerization

❑The initiation of DNA synthesis by DNA Polymerase always require

priming by a short length of RNA, called Primer.
 Three important properties of DNA Polymerase Complex
❑ Chain elongation: Chain elongation accounts for the rate (in nucleotides
per second) at which polymerization occurs. Rate of chain elongation of
DNA Pol III is 20-50 nucleotides/second.
❑Processivity: Processivity is an expression of the number of nucleotides
added to the nascent chain before the polymerase disengages from the
template. Processivity of DNA Pol III is 100 to >50,000 nucleotides.
❑Proofreading: The proofreading function identifies copying errors and
corrects them.
• Proofreading function needs 3’ to 5’ exonuclease activity
• Repair function needs 5’ to 3’ exonuclease activity
 Prokaryotic DNA Polymerase
❑Three types of Prokaryotic DNA Polymerase
• Pol I
• Pol II
• Pol III
 Bacterial DNA Polymerase
❑Main replication DNA Polymerase is DNA Polymerase III
❑DNA Polymerase with highest rate of chain elongation (Most Processive) is
Pol III
❑DNA Polymerase with proofreading activity: Pol I, Pol II and Pol III
❑DNA Polymerase with repair activity: Pol I and Pol II
❑DNA Polymerase which fills the gap in the lagging strand is Pol I
❑DNA Polymerase which polymerises Okazaki fragments is Pol III
❑DNA Polymerase which synthesizes leading strand is Pol III
❑Kornberg’s enzyme is DNAP I, as it is discovered by Arthur Kornberg
❑Arthur Kornberg described the existence of DNA Polymerase I in E coli
❑Klenow Fragment: DNA Polymerase I in which 5’ to 3’ exonuclease activity is
removed.
 Eukaryotic DNA Polymerase
❑There are Mainly five types of Eukaryotic DNA Polymerase
▪ DNAP α
▪ DNAP β
▪ DNAP γ
▪ DNAP δ
▪ DNAP ε
DNAP γ, DNAP δ, DNAP ε
have proofreading
activity
A Comparison of Prokaryotic and Eukaryotic DNA Polymerases
Steps Involved in DNA Replication in Eukaryotes
 Steps of DNA Replication
❑ Identification of the origins of replication
Fixed points on the chromosome where replication begins are called Ori
‒ In E coli; - ori C
‒ In Bacteriophage λ; - ori λ
‒ In Yeast; - Autonomous Replicating Sequence (ARS)
‒ In humans similar to Yeast
There is a Single ori in bacteria
There is Multiple ori present in eukaryotes
❑There is an AT rich sequence adjacent to ori facilitating DNA unwinding.
❑In eukaryotes ~80 bp AT rich sequence called DNA UNWINDING
ELEMENT (DUE)
Replication of DNA: origins and replication forks. A. Small, circular prokaryotic DNA. B. Long, linear eukaryotic DNA
Steps of DNA replication continued…
Ori+ ds DNA binding Protein (DNA A) opens the DNA duplex
‒ Unwinding (denaturation) of ds DNA to provide an ssDNA template
‒ Ori + ds binding protein causes local denaturation of DNA.
‒ This facilitates the further unwinding of DNA by Helicase

Role of Single Strand Binding Protein (SSB)

• Prevents the re annealing of the separated DNA strands.
• Human SSBs are called Replication Protein A (RPA)

Formation of the replication fork

‒ Unwinding of DNA forms replication bubble
‒ A pair of replication fork is replication bubble

Synthesis of RNA primer

❑by Primase that synthesizes 100–200 length Ribonucleotides

‒ DnaG is the primase in case of Prokaryotes

‒ DNAP α has primase activity in case of eukaryotes
 Replication is bidirectional
❑As the two strands unwind and separate, synthesis occurs at two replication forks that
move away from the origin in opposite directions (bidirectionally), generating a
replication bubble
 Steps of DNA replication continued…….

Initiation of DNA synthesis and elongation

❑Two strands are synthesized in different manner

1. On 3’ ----> 5’ Strand (Leading Strand) (Continuous Strand) (Forward Strand)

▪ Synthesized in continuous manner on the 3’ hydroxyl end of RNA primer in

5’ to 3’ direction
‒ In Prokaryotes by DNA Polymerase III
‒ In Eukaryotes by DNA Polymerase ε
The initiation of DNA synthesis upon a primer of RNA and the subsequent attachment of
the second deoxyribonucleoside triphosphate
Steps of DNA replication continued……….

2. On 5’----> 3’ Strand (Lagging Strand) (Discontinuous Strand) (Retrograde Strand)

▪ Synthesized in discontinuous manner.

• Small fragments of DNA are added in short spurts called Okazaki Fragment
synthesized in 5’ ----> 3’ direction.
‒ By DNAP III in prokaryotes
‒ By DNAP δ in eukaryotes
‒ Length of Okazaki fragments in Prokaryotes is 1000 to 2000 nucleotides
‒ Length of Okazaki fragments in Eukaryotes is 100 to 250 nucleotides.
 RNA primer excision and replacement by DNA
❑DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by
proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by
DNA pol I.

❑DNA pol I also has a 5′→ 3′ exonuclease activity that is able to hydrolytically remove the
RNA primer.

❑First, DNA pol I locates the space (nick) between the 3′-end of the DNA newly synthesized
by DNA pol III and the 5′-end of the adjacent RNA primer.

❑Next, DNA pol I hydrolytically removes the RNA nucleotides ahead of itself, moving in the
5′→ 3′ direction (5′→ 3′ exonuclease activity).
RNA primer excision and replacement by DNA continued…..
❑As it removes ribonucleotides, DNA pol I replaces them with
deoxyribonucleotides, synthesizing DNA in the 5′→ 3′ direction (5′→ 3′
polymerase activity).
❑As it synthesizes the DNA, it also proofreads using its 3′→ 5′ exonuclease
activity to remove errors.
❑This removal/synthesis/proofreading continues until the RNA primer is totally
degraded, and the gap is filled with DNA
❑DNA pol I uses its 5′→ 3′ polymerase activity to fill in gaps generated during
most types of DNA repair
Removal of RNA primer and filling of the resulting gaps by DNA polymerase I
Endonucleases and Exonucleases
Note;
❑Removal of RNA Primers and gap filling of the lagging strand.
‒ In Prokaryotes;—Removal of RNA Primer and Gap filling by DNA polymerase-I
‒ In Eukaryotes;- RNAse H and flap endonuclease 1 (FEN1) rather than by a DNA pol,
removes the primer
‒ DNAP δ fills the gap, where RNA Primer is removed.

❑ Sealing the nick following gap filling.

‒ DNA ligase: Seals the single strand nick between the nascent chain and Okazaki
fragments on lagging strand.
 Note;
❑Length of Okazaki fragments in Prokaryotes is 1000 to 2000 nucleotides
❑Length of Okazaki fragments in eukaryotes is 100 to 250 nucleotides
❑Time taken for replication in bacteria is 30 minutes.
❑Time taken for replication of entire human genome is 9 hours.

❑Replisome
Multimeric proteins that assemble in the replication fork are called Replisome.
It includes:
• DNA helicase
• Primase
• DNA polymerase
• Single strand binding proteins
❑Primosome
Mobile complex between helicase and primase. The primosome makes the RNA primer
required for leading-strand synthesis and initiates Okazaki fragment formation in
discontinuous lagging-strand synthesis.
 Proofreading newly synthesized DNA
❑Misreading of the templatesequence could result in deleterious, perhaps lethal,
mutations.
❑To insure replication fidelity, DNA pol III has a proofreading activity (3′→ 5′ exonuclease,)
in addition to its 5′→ 3′ polymerase activity.
❑As each nucleotide is added to the chain, DNA pol III checks to make certain the base of
the newly added nucleotide is, in fact, the complement of the base on the template
strand.
❑if the template base is C and the enzyme inserts an A instead of a G into the new chain,
the 3′→ 5′ exonuclease activity hydrolytically removes the misplaced nucleotide.
❑The 5′→ 3′ polymerase activity then replaces it with the correct nucleotide containing G
❑The 3′→ 5′ exonuclease activity removes the error in the direction opposite to
polymerization.
❑The 5′→ 3′ polymerase and 3′→ 5′ exonuclease domains are located on different
subunits of DNA pol III.
Exonuclease activity enables DNA polymerase III to proofread the newly synthesized DNA strand.
DNA replication illustration
Termination of DNA Replication

❑Replication termination in E. coli is mediated by sequence-specific

binding of the protein Tus (terminus utilization substance) to replication
termination (ter) sites on the DNA, stopping the movement of the
replication fork.
 Telomeres
▪ The ends of chromosome contain structures called telomeres
▪ Telomeres consist of short T-G repeats
▪ Human telomeres have variable number of tandem repeats of the sequence 5’ TTAGGG-3’.

They maintain the structural

integrity of the chromosome by
preventing attack by nucleases, and
allow repair systems to distinguish a
true end from a break in dsDNA
 Telomere shortening:
❑Following removal of the RNA primer from the extreme 5′-end of the lagging strand,
there is no way to fill in the remaining gap with DNA.

❑Consequently, in most normal human somatic cells, telomeres shorten with each
successive cell division

❑Once telomeres are shortened beyond some critical length, the cell is no longer able to
divide and is said to be senescent.

❑In germ cells and stem cells, as well as in cancer cells, telomeres do not shorten and the
cells do not senesce.

❑This is a result of the ribonucleoprotein telomerase, which maintains telomeric length in

these cells
Telomere and Telomerase

❑On the 5’ end of the newly synthesized linear DNA, RNA primer is
removed by RNAase H
❑This leaves a gap at the 5’ end of daughter strand.
❑In other words 3’ end of the leading strand is not synthesized.
❑This results in shortening of DNA with each cell division.
❑This is prevented by presence of Telomere and Telomerase
Telomerase (Telomere Terminal Transferase)

❑Enzyme which prevents shortening of DNA

❑Has an intrinsic RNA primer (has short piece of RNA (Terc) that acts as a template)
❑Has Reverse Transcriptase (RNA Dependent DNA Polymerase) activity
❑Present in Germ line, stem cells, most cancer cells
❑Absent from most somatic cells.
 Action of Telomerase
❑The C-rich RNA template base-pairs with the G-rich, single-stranded 3′- end of
telomeric DNA .
❑The reverse transcriptase uses the RNA template to synthesize DNA in the usual
5′→ 3′ direction, extending the already longer 3′-end.
❑Telomerase then translocates to the newly synthesized end, and the process is
repeated.
❑Once the G-rich strand has been lengthened, primase activity of DNA pol α can
use it as a template to synthesize an RNA primer.
❑The primer is extended by DNA pol α and then removed by nucleases.
Action of telomerase
 Clinical Significance of Telomerase
❑Absence of Telomerase lead to premature ageing
❑In cancer cells, there is increased Telomerase activity
❑Telomere shortening is associated with ageing, malignancy
❑Telomerase has become an attractive target for cancer chemotherapy and drug development.

❑Reverse Transcriptase

• Temin and Baltimore isolated this enzyme in 1970

• They are RNA Dependent DNA Polymerase

• Synthesize new DNA strand with RNA as template

• Thus, they reverse Central dogma of molecular genetics

• These enzymes are important in RNA Viruses like Retroviruses

• Telomerase has reverse transcriptase activity

 DNA replication inhibition by nucleoside analogs
❑DNA chain growth can be blocked by the incorporation of certain nucleoside analogs that have been
modified on the sugar portion

❑For example, removal of the hydroxyl group from the 3′-carbon of the deoxyribose ring as in 2′,3′
dideoxyinosine (didanosine), or conversion of the deoxyribose to another sugar, such as
arabinose, prevents further chain elongation.

❑By blocking DNA replication, these compounds slow the division of rapidly growing cells and viruses

❑Cytosine arabinoside (cytarabine, or araC) has been used in anticancer chemotherapy, whereas adenine
arabinoside (vidarabine, or araA) is an antiviral agent.

❑Substitution on the sugar moiety, as seen in azidothymidine (AZT), also called zidovudine (ZDV), also
terminates DNA chain elongation.

❑These drugs are generally supplied as nucleosides, which are then converted to nucleotides by cellular
kinases
 DNA REPAIR
❑DNA is subjected to a huge array of chemical, physical, and biological assaults on a
daily basis
❑ Repair of damaged DNA is critical for maintaining genomic integrity and thereby
preventing the propagation of mutations

❑The mutation can result in any of a number of deleterious effects, including loss of
control over the proliferation of the mutated cell, leading to cancer.

❑Most of the repair systems involve

▪ recognition of the damage (lesion) on the DNA,
▪ removal or excision of the damage,
▪ replacement or filling the gap left by excision using the sister strand as a template
for DNA synthesis,
▪ and ligation.

❑These excision repair systems remove one to tens of nucleotides.

Types of damage to DNA
 The mechanisms of DNA repair include

❑Nucleotide Excision Repair (NER)

❑Mismatch Repair (MMR)
❑Base Excision Repair (BER)
❑Homologous Recombination (HR)
❑Nonhomologous End-Joining (NHEJ) repair
Key concept map for DNA repair
DNA Damage, Repair Mechanisms and their Accuracy
 Mismatch Repair (MMR)
• In E. coli, mismatch repair (MMR) is mediated by a group of proteins known as the Mut proteins.
Homologous proteins are present in humans.
• MMR occurs within minutes of replication and reduces the error rate of replication from 1 in 107 to 1 in
109 nucleotides.

❑Steps involved in MMR

1. Mismatched strand identification:
• The Mut proteins that identify the mispaired nucleotide(s) discriminate between the correct strand and
the strand with the mismatch.
• In prokaryotes, discrimination is based on the degree of methylation.
• GATC sequences, which are found once every thousand nucleotides, are methylated on the adenine (A)
residue by DNA adenine methylase (DAM).
• The DNA is hemimethylated i.e. the parental strand is methylated, but the daughter strand is not.
• The methylated parental strand is assumed to be correct, and it is the daughter strand that gets repaired.
2. Mismatch Repair procedure:
▪ When the strand containing the mismatch is identified, an endonuclease nicks
the strand, and the mismatched nucleotide(s) is/are removed by an exonuclease.

▪ Additional nucleotides at the 5′- and 3′-ends of the mismatch are also removed.

▪ The gap left by removal of the nucleotides is filled, using the sister strand as a
template, by a DNA pol, typically DNA pol III.

▪ The 3′-hydroxyl of the newly synthesized DNA is joined to the 5′-phosphate of the
remaining stretch of the original DNA strand by DNA ligase.
❑ Methyl-directed mismatch repair
in Escherichia coli.
▪ Mut S protein recognizes the
mismatch and recruits Mut L.
▪ The complex activates Mut H,
which cleaves the unmethylated
(daughter) strand.
Mutations to the mismatch repair proteins lead to cancer

❑Mutation to the proteins involved in MMR in humans is associated with

hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch
syndrome.
❑Although HNPCC confers an increased risk for developing colon cancer
(as well as other cancers), only about 5% of all colon cancer is the result
of mutations in MMR.
 Nucleotide Excision Repair (NER)
• Exposure of a cell to UV radiation can result in the covalent joining of two adjacent
pyrimidines (usually thymines), producing a dimer.

• These intrastrand cross-links prevent DNA pol from replicating the DNA strand
beyond the site of dimer formation.

• Thymine dimers are excised in bacteria by UvrABC proteins in a process known as

nucleotide excision repair (NER)

• A related pathway is present in humans

• Note: Transcription-coupled repair, a type of NER, fixes DNA lesions encountered

during RNA synthesis.
 ❑Steps involved in NER
▪A UV-specificnendonuclease (called
uvrABC excinuclease) recognizes the
bulky dimer
▪ Cleaves the damaged strand on both the
5′-side and 3′-side of the lesion.
▪ A short oligonucleotide containing the
dimer is excised, leaving a gap in the DNA
strand.
▪ This gap is filled in using a DNA pol I and
DNA ligase.
Note; NER occurs throughout the cell cycle.
Pyrimidine dimers can lead to cancer
• Pyrimidine dimers can be formed in the skin cells
of humans exposed to UV radiation in unfiltered
sunlight.
• In the rare genetic disease xeroderma
pigmentosum (XP), the cells cannot
repair the damaged DNA,
• This results in extensive accumulation of
mutations and, consequently, early and numerous
skin cancers
• XP can be caused by defects in any of the several
genes that code for the XP proteins required for
NER of UV damage in humans.
 Base Excision Repair
❑DNA bases can be altered;
➢ Either spontaneously, as is the case with cytosine, which slowly undergoes deamination
(the loss of its amino group) to form uracil,
➢Or by the action of deaminating or alkylating compounds.

Examples of deaminating andalkylating compounds;

• nitrous acid, which is formed by the cell from precursors such as the nitrates,
deaminates cytosine, adenine (to hypoxanthine), and guanine (to xanthine).
• Dimethyl sulfate can alkylate (methylate) adenine.

❑Bases can also be lost spontaneously. For example, ~10,000 purine bases are lost this way
per cell per day.
❑Lesions involving base alterations or loss can be corrected by base excision repair ([BER],
 Steps involved in BER
• In BER, abnormal bases, such as uracil (which can occur in DNA by
either deamination of cytosine or improper use of dUTP instead of
dTTP during DNA synthesis) are recognized by specific DNA
glycosylases

• DNA glycosylases hydrolytically cleave them from the

deoxyribosephosphate backbone of the strand.
• This leaves an apyrimidinic site, or apurinic if a purine was
removed, both referred to as AP sites.

• Specific AP endonucleases recognize that a base is missing and

initiate the process of excision and gap filling by making an
endonucleolytic cut just to the 5′-side of the AP site.

• A deoxyribose phosphate lyase removes the single, base-free,

sugar phosphate residue.
• DNA pol I and DNA ligase complete the repair process.
 Double - strand break repair
❑Ionizing radiation, chemotherapeutic agents such as doxorubicin, and oxidative free
radicals can cause double-strand breaks in DNA that can be lethal to the cell.
❑ Such breaks also occur naturally during genetic recombination

❑dsDNA breaks cannot be corrected by the previously described strategy of excising the
damage on one strand and using the undamaged strand as a template for replacing the
missing nucleotide(s)
❑Instead, they are repaired by one of two systems.
1. nonhomologous end joining (NHEJ)
2. homologous recombination (HR),

❑ The choice between the two depends upon the phase of the cell cycle and the exact
type of DSB breaks to be repaired

❑ During the G0/G1 phases of the cell cycle, DSBs are corrected by the NHEJ pathway,
whereas during S, G2, and M phases of the cell cycle HR is utilized.
Nonhomologous end joining and Homologous recombination
• In nonhomologous end joining (NHEJ), a group of proteins mediates the recognition,
processing, and ligation of the ends of two DNA fragments.
• However, some DNA is lost in the process.
• Therefore, NHEJ is error prone and mutagenic.
• Defects in NHEJ are associated with a predisposition to cancer and immunodeficiency
syndromes.

• Homologous recombination (HR), uses the enzymes that normally perform genetic
recombination between homologous chromosomes during meiosis.
• This system is much less error prone (“error-free”) than NHEJ because any DNA that was
lost is replaced using homologous DNA as a template.

Note: Mutations to the proteins BRCA1 or BRCA2 (breast cancer 1 or 2),

which are involved in HR, increase the risk for developing breast and ovarian cancer.
Human Diseases of DNA Damage Repair
 Nucleic Acid Chemistry

CHEMISTRY OF NUCLEOTIDES

1. Explain purines and pyrimidines with suitable examples.

Purines and pyrimidines are heterocyclic compounds containing nitrogen, which form the

structure of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) (Figs 13.1, 13.2 and
Table 13.1).

Fig. 13.1: General structure of purine Fig. 13.2: General structure of pyrimidine


Table 13.1: Purines and pyrimidines
Base Purines Pyrimidines
Major bases in nucleic acids Adenine (A) Cytosine (C)
Guanine (G) Uracil (U)
Thymine (T)
Minor bases in nucleic acids 7-methyl guanine 5-methylcytosine
Dimethyl adenine 5-hydroxymethylcytosine
Metabolic intermediates and Hypoxanthine, xanthine, uric acid, caf- 5-fluorouracil
analogues feine, theophylline, allopurinol

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571

2. What are nucleosides?

Definition: Nucleosides are glycosides formed by combination of nitrogenous base with
pentose sugar (Fig. 13.3 and Table 13.2).
Composition: Base (purine or pyrimidine) + pentose sugar (ribose or deoxyribose). Ribo-
nucleoside has ribose and deoxyribonucleoside has deoxyribose.
For example: Adenine + ribose → Adenosine
Guanine + ribose → Guanosine
Uracil + ribose → Uridine
Cytosine + ribose → Cytidine
Thymine + ribose → Thymidine

Nucleic Acid Chemistry

Adenine + deoxyribose → Deoxyadenosine

Fig. 13.3: Structure of nucleoside

Table 13.2: Nucleosides

Name Examples
Nucleosides in DNA* • Deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine
Nucleosides in RNA† • Adenosine, guanosine, cytidine, uridine
Other nucleosides • Pseudouridine, thymidine, S-adenosyl methionine, 5-deoxyadenosyl
cobalamin
*
DNA, deoxyribonucleic acid; †RNA, ribonucleic acid.

3. What are nucleotides? Give some examples.

Definition: Nucleotides are phosphoric acid esters of nucleosides (nucleoside + phosphate).
They form the basic units of DNA and RNA (Fig. 13.4 and Table 13.3).
Composition: Base (purine or pyrimidine) + pentose sugar (ribose or deoxyribose) + phosphate.

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 176

 
Fig. 13.4: Structure of nucleotide (AMP)

Table 13.3: Examples and functions of nucleotides

Nucleic Acid Chemistry

Examples Functions
Adenosine triphosphate (ATP) and guanosine triphos- • High-energy compounds
phate (GTP)
Cyclic adenosine monophosphate (cAMP) and cyclic • Second messenger
guanosine monophosphate (cGMP)
3’-phosphoadenosine-5’-phosphosulfate (PAPS) • Sulfate donor
Nicotinamide adenine dinucleotide (NAD), nicotinamide • Coenzymes
adenine dinucleotide phosphate (NADP), CoASH
Uridine diphosphate (UDP)-glucose • Glycogen and UDP-glucuronic acid synthesis
UDP-glucuronic acid • Detoxification
Cytidine diphosphate (CDP)-choline • Synthesis of lecithin and sphingomyelin

Deoxynucleotides: Nucleotides with 2’-deoxyribose. For example, deoxyadenosine triphos-

phate (dATP), deoxyguanosine triphosphate (dGTP).

STRUCTURE AND FUNCTIONS OF NUCLEIC ACIDS

4. Describe Watson-Crick model of DNA with a figure. What are the differences between
DNA and RNA?
i. In 1953, Watson and Crick proposed the DNA structure (Fig. 13.5).


ii. DNA consists of two polydeoxyribonucleotide strands coiled around the same axis to


form a right-handed helix. DNA is composed of deoxyribonucleotides (deoxyribose +
phosphate in diester linkage + bases like A, T, C, G).
iii. Polydeoxyribonucleotide strand is formed by phosphodiester bond between 3’-OH


group of one sugar and 5'-OH group of another sugar.

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771

iv. One strand is oriented in 5’ to 3’ direction; the other is oriented in 3’ to 5’ direction

(antiparallel).
v. The two strands are complementary to each other and are held together by hydrogen
bonds between the bases.
vi. Each strand acts as a template for synthesis of daughter DNA strand.
vii. Base pairing rule: Adenine pairs with thymine by two hydrogen bonds; guanine pairs
with cytosine by three hydrogen bonds. Four deoxyribonucleotides are deoxyadenylate,
deoxyguanylate, deoxycytidylate and thymidylate.

Nucleic Acid Chemistry

Fig. 13.5: Structure of DNA (G, guanine; C, cytosine; A, adenine; T, thymine)

viii. Chargaff’s rule: It states that, total amount of purines are equal to total amount of
pyrimidines in a DNA double helix (A + G = T + C).
ix. The backbone of DNA is made up of alternating deoxyribose and phosphate groups
(hydrophilic). The hydrophobic nitrogenous bases are towards the core of double helix.
The bases are arranged perpendicular to the axis of helix.
x. The spatial relationship between two strands creates two types of grooves (major and
minor grooves). These grooves are the sites of interaction of DNA regulatory proteins.
xi. The diameter of the helix is 2 nm (20 Å).
xii. Each turn of the helix has 10 base pairs with a pitch of 3.4 nm (34 Å) and the bases
are 0.34 nm (3.4 Å) apart from each other along the helix.

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 178

 
Types of DNA
• B-form—DNA usually seen in human cells
• A-form—right-handed helix with 11 base pairs per turn
• Z-form—left-handed helix with 12 base pairs per turn.
Differences between DNA and RNA are given in Table 13.4.

Table 13.4: Differences between DNA and RNA
DNA RNA
Found in nucleus Found in nucleus and cytoplasm
Bases are A, T, G and C Bases are A, U, G and C
Nucleic Acid Chemistry

Deoxyribose is the sugar component Ribose is the sugar component

Double stranded Single stranded (usually)
Genetic information is transcribed to form different Genetic information is translated to form proteins
types of RNA

5. Describe the structure and functions of different types of RNAs.

Ribonucleic acid (RNA) is a single-stranded polymer of ribonucleotides linked by phos-

phodiester bond between 3’-OH of a preceding nucleotide and 5’-OH of next nucleotide.
Ribonucleotides contain three major components:

i. Ribose sugar.


ii. Nitrogenous bases—purines (adenine and guanine) or pyrimidines (cytosine and uracil).
iii. Phosphate.


Types
Messenger RNA
• Single-stranded polyribonucleotide strand formed by transcription. It carries genetic infor

­
mation from DNA for protein synthesis [heterogeneous RNA (hnRNA), the precursor form
of messenger RNA (mRNA), is processed to form mRNA]
• The mRNAs differ in size and sequences depending upon the protein that has to be syn-

thesized (heterogeneous)
• The coding region of mRNA is sandwiched between initiator codon (AUG) and terminator

codons (UGA, UAA, UAG)

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• 5’ cap: The eukaryotic mRNA is capped at the 5’ end by 7-methyl guanosine triphosphate,
which protects it from hydrolysis by 5’ exonuclease; it also helps in initiation of protein
synthesis
• Poly(A) tail: 3’ terminal contains a polymer of adenylate residues, which stabilizes the mRNA
• The mRNA is complementary to template strand of DNA.
Transfer RNA (Fig. 13.6)
i. Transfer RNA (tRNA) functions as an adapter, which brings a specific amino acid from
cytosol to the site of protein synthesis.
ii. It is small in size (75 nucleotides).
iii. Although there are 20 amino acids, around 32 tRNAs are found in humans.

Nucleic Acid Chemistry

iv. Intrastrand hydrogen bonds present in tRNA gives it a clover leaf shape.
v. A highly conserved sequence CCA is present towards the 3’ end (acceptor arm). The last
nucleotide, adenine at the 3’ end is involved in binding covalently to a specific amino acid.
vi. Three loops present in tRNA are:
• D arm: Formed by 2 or 3 dihydrouridine residues
• Anticodon arm: It has a triplet codon (complementary to the codon on mRNA molecule)
that specifically interacts with the codon on mRNA
• TΨC arm: T, Ψ and C stands for thymine, pseudouridine and cytosine.

Fig. 13.6: Structure of tRNA

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Ribosomal RNA
i. Ribosomal RNAs (rRNAs) are the most abundant forms of RNA, which are associated with


ribosomes.
ii. They have catalytic activity (like enzymes). They have a role in translation. Ribosomal


subunits are given in the Table 13.5.
Table 13.5: Subunits of ribosome
Subunits Prokaryotes Eukaryotes
Larger subunit 50S 60S
Smaller subunit 30S 40S
Nucleic Acid Chemistry

Key Points
Nucleoside: Base (purine or pyrimidine) + pentose sugar (ribose or deoxyribose).

Nucleotide: Base (purine or pyrimidine) + pentose sugar (ribose or deoxyribose) + phosphate.

Purine analogues: Allopurinol and 6-mercaptopurine used in the treatment of gout and cancer

respectively.
Pyrimidine analogues: 5’-fluorouracil (thymidylate synthase inhibitor) used in the treatment of cancer.

Nucleoside analogues: Arabinosylcytosine and 5’-iododeoxyuridine are used in the treatment of

cancer and herpetic keratitis respectively.
Base pairing rule: Adenine-thymine is linked by two hydrogen bonds and guanine-cytosine are held

together by three hydrogen bonds.
Chargaff's rule: Total amount of purines are equal to total amount of pyrimidines in double helix

(A + G = T + C).
Melting temperature (Tm) for DNA: At 90oC, half of double-stranded DNA denatures into single-

stranded DNA.
Small nuclear RNA (snRNA): The snRNAs U 1, U2, U4, U5, U6 are required for splicing of hetero-

geneous nuclear RNA.
Unusual bases and nucleosides in the tRNA: Thymidine, dihydrouracil, hypoxanthine, pseudouridine.

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 Molecular Biology-I

REPLICATION

1. Write an account on replication. Add a note on xeroderma pigmentosum.

Definition: It is the process in which two identical copies of daughter deoxyribonucleic
acid (DNA) molecules are formed from parent DNA during cell division.
Replication is semiconservative: Each daughter DNA gets one strand from parent DNA.
The other strand is newly synthesized.
Requirements for DNA replication

i. Double-stranded DNA.


ii. Deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxythy-


midine triphosphate (dTTP) and deoxycytidine triphosphate (dCTP).
iii. Enzymes:

• DNA helicases: Unwind the double helix using energy (ATP)
• DNA topoisomerase I (nuclease and ligase activity): Relieves the supercoiling of DNA
by cutting and resealing one strand of DNA
• DNA topoisomerase II (nuclease and ligase activity): Relieves the supercoiling of DNA
by breaking and resealing both the strands of DNA
• DNA polymerase: It synthesizes a complementary strand of DNA from single strand
of DNA (template) by polymerization of deoxynucleotides
– DNA polymerase I: Required for proofreading and fills the gap between Okazaki frag-


ments of lagging strand; in proofreading, copying errors are identified and corrected
(5’-3’ polymerase activity, 3’-5’ and 5’-3’ exonuclease activity)
– DNA polymerase II: Required for proofreading and DNA repair


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– DNA polymerase III: It is the main replication enzyme, also required for proofreading
(5’-3’ polymerase activity and 3’-5’ exonuclease activity).
• DNA ligase: Seals the nick in single strand as well as in Okazaki fragments.
iv. Proteins:
• dnaA: Binds to AT-rich region → unwinding of DNA
• Single-stranded DNA-binding proteins (SSB proteins): Prevent annealing of separated
DNA strands
• Primase: Initiates synthesis of short segment of RNA (RNA primer).

Steps in DNA Replication

i. Identification of site of origin of replication (ori) by specific proteins: Origin of replica-

Molecular Biology-I
tion is a unique nucleotide sequence from where DNA replication begins. In prokaryotes,
there is a single ori, which consists of AT-rich region. In eukaryotes, multiple origins of
replication exist.
ii. Unwinding of double-stranded DNA to form 2 single-stranded DNA (ssDNA): dnaA
protein binds to AT-rich region in the origin of replication → local melting/unwinding
of dsDNA → short segment of ssDNA formed (required for initiation of DNA synthesis).
Single strand binding (SSB) proteins attach to each strand and prevent their annealing
(maintain DNA in single strand form). Separation of strands of DNA is required as DNA
polymerase binds to single strand of DNA.
iii. Formation of replication fork, synthesis of primer and initiation of DNA synthesis (Fig
15.1): Unwinding of DNA causes formation of replication fork → DNA helicase binds to the
fork → unwinding of adjacent double-stranded region → primase binds to DNA at 3’ end
of each strand → synthesis of short segment of RNA (RNA primer) → DNA polymerase
using RNA primer, initiates synthesis of daughter strand (complementary to template) in 5’
→ 3’ direction. Both parent strands are simultaneously replicated in the 5' → 3' direction.
The replication forks, thus, advance in opposite direction from their origin. This process
results in the formation of 'replication bubbles'.
Direction of DNA synthesis
a. Leading strand: DNA is synthesized continuously in 5’ → 3’ direction towards the
replication fork (Fig. 15.1) and it needs only one RNA primer.
b. Lagging strand: It is synthesized in short stretches in 5’ → 3’ direction, but away from
replication fork. These short stretches of discontinuous DNA are called Okazaki frag-
ments. It requires many RNA primers.

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Molecular Biology-I

Fig. 15.1: Replication fork (DNA, deoxyribonucleic acid; SSBs, single-stranded DNA-binding proteins;
RNA, ribonucleic acid).

Unwinding of DNA during replication creates supercoils, which are relieved by topoi-


somerase I and II.

iv. Elongation


a. It is the process where there is sequential addition of deoxynucleotides via phosphodiester


bonds. First phosphodiester bond is formed between 3’–OH group of RNA primer and 5’
phosphate group of first entering deoxynucleotide. DNA polymerase (DNA pol III) elon-
gates new DNA strand by adding deoxynucleotides (dATP, dGTP, dTTP, dCTP) one at a
time, to the 3’ end of growing chain, complementary to bases in the template strand.
b. Proofreading of newly formed DNA: Any errors due to mismatched nucleotides during


replication are immediately repaired to prevent lethal mutations. This is mainly done by
DNA polymerase III and DNA polymerase I.
c. Excision of RNA primer.


• DNA polymerase I, which removes the RNA primer (5’-3’ exonuclease activity) and
synthesizes DNA that replaces RNA using 5’-3’ polymerase activity; it also proofreads
the new chain using 3’-5’ exonuclease activity

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193

• DNA ligase: Catalyzes the formation of phosphodiester bond between DNA syn-
thesized by DNA polymerase III and that formed by DNA polymerase I using
energy (ATP).
v. In eukaryotes, an additional step occurs. The newly synthesized dsDNA reforms chromatin
structure.

Xeroderma Pigmentosum
Xeroderma pigmentosum is an autosomal recessive disorder with defective nucleotide ex-
cision repair due to deficiency of enzyme ultraviolet (UV) specific endonuclease. In this
condition, exposure to UV light can cause mutations in DNA (pyrimidine dimers), which may
lead to cancer.

Molecular Biology-I
2. What are Okazaki fragments?
Okazaki fragments are short segments of single-stranded DNA that are synthesized dis-
continuously on lagging strand during DNA replication (refer Fig. 15.1). DNA polymerase
I removes the RNA primers (5’-3’ exonuclease activity) between these fragments and syn-
thesize DNA that replaces RNA (5’-3’ polymerase activity). DNA ligase finally joins these
fragments (refer Elongation, p. 192).
3. Write a note on inhibitors of replication.
Inhibitors of replication, mechanism of action and therapeutic uses are given in Table 15.1.
Table 15.1: Inhibitors of replication
Inhibitor Mechanism of action Therapeutic use
Ciprofloxacin, novobiocin, nalidixic acid DNA gyrase inhibition Antibiotics
Etoposide, doxorubicin Inhibit topoisomerase II Anticancer agents
Cytosine arabinoside Prevents chain elongation Anticancer agent
Adenine arabinoside Prevents chain elongation Antiviral drug

Key Points
DNA replication occurs in S (synthetic) phase of cell cycle.
Proofreading: It is mainly done by DNA polymerase III and DNA polymerase I.
RNA primer (5–12 nucleotides long): Required for DNA synthesis; produced by primase.
Postreplicative modifications of DNA: These include methylation of DNA and mismatch repair.
Huntington's disease: Autosomal dominant disease with motor and cognitive dysfunction. This is
due to expansion of trinucleotide repeats (CAG), which codes for glutamate in huntingtin protein
(altering its function).

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Xeroderma pigmentosum: It is an autosomal recessive disorder with defective nucleotide excision

repair due to deficiency of enzyme UV-specific endonuclease.
Fanconi anemia: Due to defective repair of interstrand DNA cross-links presenting with micro cephaly,

­
mental retardation, anemia and leukopenia.
Telomeres: It is the 3' end of mammalian DNA with 5'-TTAGGG-3' repeats. The number of these

repeats decreases with each cell division and indicates normal aging of cells. In germ cells, the
enzyme telomerase maintains the length of telomere.
Cancer and telomerase: Telomerase activity is found to be high in cancer cells, which make them

immortal and replicate indefinitely. Telomerase inhibitors are under development as potential anti-
cancer agents.

TRANSCRIPTION
Molecular Biology-I

4. Write briefly about promoter regions.

Promoter region: These are highly conserved regions of DNA both in pro- and eu-karyotes,

which are recognized by RNA polymerases to initiate the process of transcription.

Prokaryotic Promoters (Fig. 15.2)

a. Pribnow box (TATA box): Stretch of 6 nucleotides (5’-TATAAT-3’) located about 10 nucle-

otides upstream from transcription start site. This has low melting temperature due to lack
of GC pairs. This will result in unwinding of DNA.
b. –35 sequence: It is a sequence of 8 nucleotides (5’-TGTTGACA-3’) located approximately

35 bp upstream from transcription start site.

Fig. 15.2: Prokaryotic promoter regions

Eukaryotic Promoters
About 25 nucleotides upstream of the transcription start site, similar to prokaryotes, a TATA
or Hogness box is present. Another consensus sequence CAAT box is present about 70–80
nucleotides upstream of transcription start site.

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195

All these promoters help in deciding the origin of transcription by facilitating effective
binding of RNA polymerase.
5. Describe transcription process. Add a note on inhibitors of transcription.
Definition: Transcription is the process of synthesizing RNA from a DNA template. It takes
place in the 5' → 3' direction. The newly synthesized mRNA [primary transcript, heteroge-
neous nuclear RNA (hnRNA)] is identical to the other DNA strand—the coding strand (except
having uracil in place of thymine).

Requirements
• DNA to be copied
• RNA polymerase, RNAP (holoenzyme): Core enzyme (2α, 1β, 1β') + σ (sigma) factor. It

Molecular Biology-I
is the enzyme that synthesizes mRNA
• Termination factor—ρ (rho): For termination of transcription
• ATP
• Helicase: Unwinding of DNA during transcription
• Topoisomerase I and II: Remove supercoiling.

Steps in Transcription
Steps in transcription include initiation, elongation and termination.
i. Binding of RNAP to the template strand of DNA and formation of preinitiation complex:
RNA polymerase (holoenzyme) binds to promoter region (-35 sequence) of DNA. It then
moves on and binds to TATA box → causes local unwinding of DNA.
ii. Initiation of chain synthesis: The first nucleotide of RNA binds to nucleotide binding site
of 'b' subunit of RNA polymerase to form 5’ end of RNA. RNA polymerase moves to next
base on the template strand. A corresponding nucleotide binds to RNA polymerase and
phosphodiester bond is formed between the two nucleotides (Fig. 15.3).
iii. Clearance of promoter: Nucleotides continue to be added. Once RNA has 10–20 nucle-
otides, RNA polymerase leaves the promoter site (promoter clearance) and moves along
template strand.
iv. Elongation: After promoter clearance, elongation phase starts. New nucleotides are added to
the nascent mRNA complementary to the template strand. RNA polymerase uses ribonucle-
otides ATP, GTP, CTP and UTP. For addition of each ribonucleotide, energy equivalent to
two ATP is used (Table 15.2). Elongation complex containing RNA polymerase moves along
DNA template → unwinding of DNA downstream by RNAP.

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196 Quick Review of Biochemistry for Undergraduates

 
Fig. 15.3: Transcription
Molecular Biology-I

Unwinding causes supercoils, which is relieved by topoisomerases.

Table 15.2: DNA template is copied in the following manner
DNA template Corresponding base on RNA strand
G C
C G
T A
A U
v. Termination: Process of transcription continues until termination signal sequence is reached


on the template strand of DNA.
Rho-dependent termination: Rho is a specific protein, which recognizes the termination signal

→ dissociation of RNAP from DNA and release of newly synthesized mRNA.
Rho-independent termination: Newly synthesized RNA forms hairpin structure. It also has

series of Us near the 3' end, which helps in release of newly synthesized mRNA.
The inhibitors of transcription are given in Table 15.3.

Table 15.3: Inhibitors of transcription
Drug Mechanism of action Application
Rifampicin Inhibits ' ' subunit of prokaryotic RNA poly- Treatment of tuberculosis
b
merase
Actinomycin D Binds to DNA template and interferes with move- Anticancer agent
ment of RNA polymerase
α-amanitin Inactivates RNA polymerase II Mushroom poison

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197

6. Write a note on histones.

• Histones are proteins, rich in lysine and arginine; they are associated with chromatin
• They form ionic bonds with DNA
• They are required for packaging of DNA into nucleosomes
• They play an important role in regulation of gene expression
• They are of five types namely H1, H2A, H2B, H3 and H4
• Histone acetylation/deacetylation can alter their interaction with DNA. This makes the
chromatin more/less accessible for transcription, respectively.
7. Write briefly on reverse transcriptase.
Definition: Reverse transcriptase is the RNA-dependent DNA polymerase. It synthesizes

Molecular Biology-I
DNA from RNA. It is present in retroviruses where it synthesizes viral DNA from viral
RNA (Fig. 15.4).

Fig. 15.4: Synthesis of viral proteins by reverse transcriptase

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Applications

• Preparing complementary DNA (cDNA) library

• RT-PCR (reverse transcriptase-polymerase chain reaction)
• Reverse transcriptase inhibitors: zidovudine, lamivudine, etc. are used in the treatment of
HIV infection.
8. Write a note on post-transcriptional modification.
Definition: Process by which newly synthesized RNA (primary transcript) is modified to

mature mRNAs, rRNAs and tRNAs. This type of modification is mainly seen in eukaryotic
mRNA, pro- and eukaryotic rRNA and tRNA.
Molecular Biology-I

Post-transcriptional Modifications of mRNA

a. 5’ capping: The primary RNA transcript is known as hnRNA. 7-methylguanosine triphos-

phate is attached to the 5’ end of this RNA (capping). S-adenosyl methionine is the methyl
donor. 5’ capping helps in stabilization of mRNA and initiation of translation.
b. Poly-A tail: A chain of 20–250 adenine nucleotides are attached to the 3’ end of mRNA.

Poly-A tail helps in stabilization of RNA and translation.
c. Removal of introns: The primary transcript has exons (coding region) and introns (non-

coding regions). Small nuclear RNAs (snRNAs) in association with some proteins form
small nuclear ribonucleoprotein particles (SnRNPs) → bind to RNA to form spliceosomes,
which remove the introns and join the exons (Fig. 15.5).

Fig. 15.5: Removal of introns of mRNA

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Post-transcriptional Modifications of tRNA (Pro- and Eu-karyotes)

• CCA tail added to 3’ end
• Some bases are modified by reduction, methylation, deamination, etc.

Post-transcriptional Modifications of rRNA (Prokaryotes)

• 23S rRNA + 5S rRNA + proteins → 50S subunit
• 16S rRNA + proteins → 30S subunit; 30S + 50S → 70S ribosome.

Post-transcriptional Modifications of rRNA (Eukaryotes)

• 28S rRNA + 5.85S rRNA + 5S rRNA + proteins → 60S subunit

Molecular Biology-I
• 18S rRNA + proteins → 40S subunit; 40S + 60S → 80S ribosome.

Key Points
RNA polymerase has no proofreading activity, so transcription is more error prone compared to
replication.
Mushroom poisoning (α-amanitin): Inhibits RNA polymerase II and prevents elongation.
Spliceosome: It removes the introns and joins the exons to form mature mRNA.
Reverse transcriptase: It is the RNA-dependent DNA polymerase present in retroviruses, where
they copy the viral RNA genome into DNA.

TRANSLATION

9. Describe the process of translation in a. Prokaryotes b. Eukaryotes.

Definition: Translation is the process by which mRNA is translated to produce a polypep-
tide sequence, also known as a protein (Fig. 15.6).

Fig. 15.6: Synthesis of proteins

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Requirements for Translation
a. Amino acids: For synthesis of polypeptide chain.

b. Transfer RNA: At least one tRNA per amino acid.

c. Aminoacyl-tRNA synthetase: Required for attachment of amino acids to the specific tRNAs.

This requires energy (two ATP molecules).
d. Messenger RNA (mRNA): Has codons, which dictate synthesis of polypeptide chain.


e. Ribosomes (50S, 30S in prokaryotes; 40S, 60S in eukaryotes): Located in the cytosol as

free form or associated with endoplasmic reticulum.
• A, P and E sites on ribosome (p. 201) for binding tRNA
– A-site: For binding of incoming aminoacyl-tRNA as specified by codon at that site


Molecular Biology-I

– P-site: Has peptidyl tRNA (tRNA with newly synthesized chain of amino acids)


attached to it
– E-site: It is occupied by empty tRNA.


f. Initiation, elongation and termination factors.


g. Energy from:

• 2 ATP for binding of amino acids to specific tRNA
• 2 GTP for binding of aminoacyl-tRNA to A-site and translocation.
h. Binding between codon on mRNA and specific anticodon of tRNA: As per base pairing

rule (Fig. 15.7).

Fig. 15.7: Codon-anticodon interaction

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201

Steps of Translation in Prokaryotes

i. Initiation: Mechanism by which ribosome recognizes nucleotide sequence on mRNA to

initiate the translation.
• Shine-Dalgarno sequence (purine-rich region 5’-UAAAGGAGG-3’): Located 6–10 bases
upstream from AUG codon on the mRNA; this facilitates the binding of 30S ribosome
to mRNA
• Initiation codon AUG (Fig. 15.8): Located on mRNA; it is recognized by initiator tRNA
with N-formylmethionine.
Transformylase
tRNA-methionine + N10 formyl THF tRNA-N-formylmethionine
(fMet-tRNA) + THF

Molecular Biology-I
a. Formation of 30S initiation complex: 30S ribosome + mRNA + aminoacyl-tRNA
specified by start codon + initiation factors (IF 1, 2, 3) → 30S initiation complex is
formed (Fig. 15.8).
b. Formation of 70S initiation complex (Fig. 15.9):
IF 1, 2, 3
50S ribosome + 30S initiation complex + GTP 70S initiation complex

The fMet-tRNA is at the P-site.

Fig. 15.8: 30S initiation complex Fig. 15.9: 70S initiation complex

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ii. Elongation:


a. Binding of appropriate tRNA to empty A-site: Elongation factors (EF-Tu, EF-Ts), in the


presence of GTP, will help in binding of appropriate tRNA with an amino acid to next
codon in the empty A-site (Fig. 15.10).
Molecular Biology-I

Fig. 15.10: Binding of tRNA to A-site

b. Peptide bond formation: Peptidyl transferase transfers the amino acid/peptide from the

P-site on to amino acid at the A-site and catalyzes peptide bond formation between the
amino acids. The tRNA at the P-site now does not have an amino acid (empty tRNA).
c. Translocation: Ribosome moves a distance of three nucleotides along mRNA in the

5’-3’ direction in the presence of GTP and EF-G. Thus, the empty tRNA, which was
at the P-site, now lies at E-site; peptidyl tRNA at A-site is now at P-site; A-site is
empty (Fig. 15.11).

Fig. 15.11: Translocation

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203

A new aminoacyl-tRNA binds to corresponding codon on mRNA at A-site. These

steps for formation of required peptide are repeated until termination codon of mRNA
is at A-site.
iii. Termination: When termination codon (UAA, UGA, UAG) of mRNA is at A-site, it is rec-
ognized by release factor (RF). RF binds to this site and releases newly formed peptide in
the presence of GTP. The ribosome dissociates into its subunits.

Steps of Translation in Eukaryotes

i. Formation of aminoacyl-tRNA:
Aminoacyl-tRNA synthetase
Amino acid + ATP + tRNA Aminoacyl-tRNA + AMP

Molecular Biology-I
ii. Initiation of protein synthesis:
a. Dissociation of ribosome → 40S subunit + 60S subunit.
b. Formation of 43S preinitiation complex:
GTP + initiation factor 2 + Met-tRNA (methionine is the first amino acid to be incorpo-
rated in protein synthesis)

Complex formed

Binds to 40S ribosomal subunit

43S preinitiation complex formed
c. Formation of 48S initiation complex:
mRNA binds to 43S preinitiation complex

48S initiation complex
The initiation (start) codon AUG on mRNA is identified by Kozak sequence.
The anticodon of Met-tRNA binds to initiation codon AUG on mRNA.
d. Formation of 80S initiation complex:
48S initiation complex binds with 60S ribosomal subunit

80S initiation complex formed
Met-tRNA is attached to P-site of ribosome. The A and E sites are free.

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iii. Elongation: Addition of amino acids to the new growing peptide chain.


a. Binding of new, appropriate aminoacyl-tRNA to codon at acceptor A-site.


b. Formation of peptide bond by peptidyl transferase between new amino acid of tRNA


at A-site and amino acid attached to tRNA at P-site → transfer of amino acid/growing
peptide chain from P-site to amino acid at A-site. Thus, tRNA at P-site now does not
have any amino acid.
c. Translocation:


Ribosome moves along mRNA by 3 nucleotides (one codon) in the 5’ → 3’ direction


Movement of tRNA from P to E site from which it is released

Movement of peptidyl-tRNA from A to P site.

A-site is empty—a new aminoacyl-tRNA binds with codon of mRNA at this site.
Molecular Biology-I

iv. Termination:


Process of addition of amino acids continues till the termination (stop) codon on mRNA is

reached at the A-site. There is no tRNA with anticodon complementary to the stop codon
at A-site.
Releasing factor recognizes stop codon at A-site



Binds to A-site


Causes release of newly formed peptide chain, tRNA and mRNA from ribosomes


Dissociation of 80S ribosome → 60S + 40S subunits

10. Describe the properties of genetic code. Add a note on Wobble hypothesis.

• Genetic code is a sequence of 3 nucleotides (triplet code) in mRNA coding for an amino acid
• There are totally 64 different codons (43) from 4 nucleotides in different combinations
• AUG: Start codon, codes for methionine
• UAA, UAG, UGA: Stop codons, do not code for any amino acid—cause termination of
peptide synthesis.

Properties of Genetic Code

• Universal: Same codons code for same amino acids in all living organisms
• Specific: The specific codon always code for the same amino acid

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205

• Non-overlapping and comma less: Codons are read in a continuous manner without any
punctuation
• It is degenerate (redundant): An amino acid may have more than one codon coding for
it, since there are 61 codons for 20 amino acids, e.g. arginine has six codons.

Wobble Hypothesis
Pairing between the bases at third position (last nucleotide) of codon on mRNA and first
position of anticodon of tRNA is non-traditional (not always as per Watson-Crick rule). So,
a single tRNA anticodon can bind to more than one codon (refer Fig. 15.7). For example,
the codons for glycine GGU, GGC and GGA pair with a single anticodon CCI of tRNA. The
base I at third position of anticodon can pair with either U, C or A of mRNA codon (Table
15.4). This is 'Wobble' [Note: Pairing between bases at first and second position of codon and

Molecular Biology-I
that of anticodon (second and third position) is as per Watson-Crick rule].
Table 15.4: Non-traditional base pairing between codon and anticodon
mRNA codon (5' → 3') for glycine GGU GGC GGA
tRNA anticodon (3' → 5') for glycine CCI CCI CCI

11. Write a note on inhibitors of translation.

Inhibitors of translation and clinical significance are given in Table 15.5.
Table 15.5: Inhibitors of translation
Inhibitor Site of action Clinical significance
Streptomycin Binds to 30S subunit and interferes with ini- Antibacterial agent
tiation
Tetracycline Inhibits binding of aminoacyl-tRNA to m-RNA Antibacterial agent
ribosome complex
Chloramphenicol Inhibits peptidyl transferase Antibacterial agent
Erythromycin Inhibits translocation Antibacterial agent
Diphtheria toxin Inactivates elongation factor (eEF-2) and pre- Causes diphtheria
vents translocation
Puromycin Structural analogue of tyrosinyl-tRNA and binds Antibiotic used in cell cultures
to A-site causing premature release of poly-
peptide chain

12. Write a short note on post-translational modification.

Definition: Process by which newly synthesized polypeptide chain undergoes modifications
to produce biologically active protein.

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i. Trimming: Proteins are synthesized as functionally inactive large precursors. They undergo

trimming by proteases to produce functionally active protein. For example,
Proinsulin Insulin

Pepsinogen Pepsin

Trypsinogen Trypsin

ii. Covalent modification: Proteins may be activated or inactivated by covalent attachment

of variety of chemical groups.
a. Phosphorylation: Hydroxyl group of serine, threonine and tyrosine residues in protein

can undergo phosphorylation.
Phosphorylase kinase

Molecular Biology-I

Glycogen phosphorylase Glycogen phosphorylase (P)

(inactive) (active)



b. Glycosylation: Attachment of carbohydrate to hydroxyl groups of serine, threonine,

hydroxylysine (O-linked) and asparagine (N-linked).
­
Collagen synthesis: Hydroxylysine undergo glycosylation.

c. Hydroxylation: Proline and lysine undergo hydroxylation during collagen synthesis.

d. Carboxylation: Clotting factors undergo g-carboxylation of glutamic acid residues in the

presence of vitamin K during coagulation.
e. Farnesylation: For anchoring of proteins to membranes.

Key Points
Starting codon: AUG (methionine).

UAA, UAG, UGA: Stop codons, do not code for any amino acid.

Selenocysteine: It is considered as 21st amino acid, which is coded by UGA stop codon.

Protein farnesyltransferase inhibitors: Tipifarnib; anticancer agent used in the treatment of acute

myeloid leukemia.
Protein targeting: Is sorting of synthesized protein into required location.

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 Molecular Biology-II

DNA REPAIR AND MUTATIONS

1. Explain causes and types of DNA damage.

Causes of DNA Damage

• Mismatch of bases during replication
• Spontaneous deamination: Changes the cytosine (C) to → uracil (U), adenine (A) → hypo­
xanthine (H) and guanine (G) → xanthine (X)
• Physical agents: Ultraviolet (UV) light, X-ray, ionizing radiation
• Chemical agents (mutagens): For example, anticancer drugs like methotrexate, cyclophos­
phamide, etc.

Types of DNA Damage

• Single base alteration: Nucleotide deletion/insertion, loss of amino groups from C, A, G,
alkylation of base, base analogue incorporation
• Two base alteration: Thymine dimer formation by cross-linkage between bases of same
strand (induced by UV radiation)
• DNA single/double strand breaks: Induced by ionizing radiation/reactive oxygen species.
2. Explain various DNA repair mechanisms and add a note on xeroderma pigmentosum.
In order to maintain the integrity of the genome, DNA undergoes repair.

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Nucleotide Excision Repair (NER)
For example, repair of pyrimidine dimers and base adducts (Fig. 16.1). UV specific endonu­

-
clease recognizes and cleaves a piece of DNA on both ends of the dimer → DNA polymerase
and DNA ligase together will repair and replace the gap with proper nucleotides. Defects in
this type of repair mechanism can lead to xeroderma pigmentosum, Cockayne's syndrome,

­
ataxia telangiectasia, etc.
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­
Molecular Biology-II

Fig. 16.1: Nucleotide excision repair

Xeroderma Pigmentosum
Refer to Chapter 15, question number 1.

Mismatch Repair
i. Replication errors (escaped from proofreading) → mismatch of one to several bases (e.g.
instead of thymine, cytosine may be incorporated opposite to adenine).
Base mismatch on the daughter strand is recognized by endonuclease → cleavage of

strand near the defect → mismatched bases are removed from the daughter strand by
exonucleases → DNA polymerase I and DNA ligase repair and replace the gap with
appropriate nucleotides (Fig. 16.2).
ii. Defect in mismatch repair: May lead to hereditary non polyposis colon cancer.
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Ch-16.indd 208 21-06-2014 11:52:07
  

Molecular Biology-II
Fig. 16.2: Mismatch repair

Base Excision Repair

Cytosine, adenine and guanine can undergo spon­
taneous deamination to form uracil, hypoxanthine
and xanthine respectively. Abnormal bases are recog­
nized by DNA glycosylases → hydrolytically remove
the abnormal base from their sugar → apyrimidinic
or apurinic site (AP site) → AP endonucleases →
produce break in the strand → deoxyribose phos­
phate lyase → removes sugar phosphate residue
→ DNA polymerase I and ligase → regeneration of
strand (Fig. 16.3).

Double-strand Break Repair

Double-strand break repair is caused by ionizing
radiation, free radicals and chemotherapy.

Repair Mechanism
• Non-homologous end joining (NHEJ): Two non-
homologous ends of the broken DNA strand are
brought together by proteins with some loss of
DNA Fig. 16.3: Base excision repair

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• Homologous recombination: Two non homologous ends of the broken DNA strand are

-
brought together by proteins without loss of DNA.
3. Define and classify mutations with examples.
Definition: A mutation is a change in the sequence of nucleotides of DNA. It may or may

not alter the amino acid sequence of the protein encoded by that gene.

Chromosomal Mutations
• Philadelphia chromosome: Chromosomes 9–22 translocation → activation of c Abl; seen

-
in 80% of chronic myeloid leukemia cases
• Burkitt's lymphoma: 8–14 translocation → c Myc activation
Molecular Biology-II

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• Non-Hodgkin's lymphoma: 14–18 translocation → activation of bcl 2 → suppression of

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apoptosis.

Gene Mutation
• Point mutation
• Frameshift mutation
• Others.
Point mutation: Alteration in a single base is point mutation. This is of two types depend­
ing on the type of base replaced.
a. Transition: Purine replaced by purine or pyrimidine replaced by pyrimidine.
For example, A ↔ G or C ↔ T.
b. Transversion: Purine replacing pyrimidine or pyrimidine replacing purine.
For example, A ↔ T or C ↔ G or A ↔ C or T ↔ G.
Effects of Point Mutation
a. Silent mutation: New codon formed after mutation codes for the same amino acid as


original codon. There is no change in the amino acid sequence and property of protein.
b. Missense mutation: New codon formed after mutation codes for a different amino acid.

This alters the amino acid sequence and may alter the function of protein. The effects
could be acceptable (function of protein not altered), partially acceptable (protein function
is partially affected) and non acceptable (non functional protein formed—could be fatal)
-
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(Table 16.1).

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c. Nonsense mutation: New codon formed after mutation codes for terminator codon leading
to premature termination of protein synthesis (Fig. 16.4).

Fig. 16.4: Various consequences of point mutations

Table 16.1: Effects of missense mutations (A, B = 'α' helical segments of globin chain)

Molecular Biology-II
Effects Normal hemoglobin Mutated hemoglobin
Acceptable B 61: AAA (lysine) B 61: AAU (asparagine): Hb Hikari
Partially acceptable B 6: GAG (glutamic acid) B 6: GUG (valine): Hb S (sickle cell disease)
Non-acceptable A 58: CAC (histidine) A 58: UAC (tyrosine): Hb M

Frameshift mutations: It occurs due to insertion or deletion of nucleotides. It has more

serious consequences than point mutation as entire frame (sequence of codons for amino
acids) changes from the point of insertion or deletion. It can lead to formation of a non-
functional protein, premature termination of protein synthesis (Fig. 16.5, p. 212).
The amino acid sequence, distal to insertion or deletion of nucleotide, changes. For example,
frameshift mutation has been demonstrated in diseases like cystic fibrosis, thalassemia, etc.

Other Mutations
• Huntington's disease: CAG trinucleotide sequence is repeated many times → insertion of
many extra glutamine residues in the huntingtin protein → unstable proteins → accumula­
tion of protein agg­regates
• Fragile X syndrome and myotonic dystrophy: Trinucleotide expansion occurs in the un­
translated portion of a gene → ↓ in the amount of protein produced
• Splice site mutation can affect removal of introns and protein formation. For example,
β-thalassemia (incorrect splicing of β-globin mRNA) and systemic lupus erythematosus
(SLE) (autoimmune antibodies against snRNP).

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Molecular Biology-II

Fig. 16.5: Frameshift mutation

REGULATION OF GENE EXPRESSION

4. Explain Lac operon model of gene expression.

Lac Operon Model

Operon: In prokaryotes, genes that code for proteins in a metabolic pathway are arranged
in a linear fashion along with a regulatory region (Fig. 16.6) [MN: RPOS = Regulation of
Prokaryotic Operon System].
In prokaryotic operon, genes are arranged in the following order from 5’ to 3’ on the
chromosome.
• Regulatory gene: Forms mRNA → forms a repressor protein → which binds operator gene
→ prevents translation of the protein (constitutive)
• Promoter gene: RNA polymerase binds to this region
• Operator gene: Site where repressor protein binds
• Structural gene: Responsible for formation of proteins.

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Lactose Operon or Lac Operon (Fig. 16.6)

Lac operon has three structural genes coding for different enzymes required for lactose
metabolism:
• Lac Z for β-galactosidase: Hydrolyzes lactose
• Lac Y for permease: Helps in movement of lactose into cell
• Lac A for thiogalactoside transacetylase: Function unknown.
There is an operator, promoter, CAP-binding site and a regulatory gene (Lac I).
a. When Escherichia coli is exposed to glucose only, repressor protein is formed → binds
operator gene → prevents the movement of RNA polymerase (RNAP) bound to promoter
→ no proteins (enzymes) formed (repression).
b. In the presence of lactose and absence of glucose (derepression).

Molecular Biology-II
• Small amounts of lactose enter E. coli → converted to allolactose (inducer) → binds to
repressor proteins and prevents its interaction with operator gene → no obstruction for
movement of RNAP → protein (enzyme) synthesis occurs
• Absence of glucose → (+) adenylyl cyclase → ↑ cyclic adenosine monophosphate (cAMP) →
cAMP-catabolite activator protein (CAP) complex → binds to CAP site → ↑ ability of bound
RNAP to initiate transcription of lac Z, lac Y and lac A genes to produce β-galactosidase,
permease and thiogalactoside transacetylase, respectively. These enzymes help E. coli
to metabolize lactose to produce energy.

Fig. 16.6: Regulation of prokaryotic Lac operon system [MN: RPOS] (RNAP, ribonucleic acid polymerase)

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c. When both glucose and lactose are available:
Operator site is free from inhibition as allolactose binds with repressor protein.
Glucose → ↓ cAMP → no CAP cAMP complex formation → RNAP cannot function

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effectively → no protein (enzymes) formed → lactose cannot be utilized. Hence, glucose
is used first even though operator site is free. After all glucose is utilized by bacteria → ↑
cAMP → cAMP CAP complex formation → (+) RNAP → protein (enzyme) formation.
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5. Briefly explain eukaryotic gene regulation.
Unlike prokaryotes, eukaryotic gene regulation is much complex. It can occur by six dif­
ferent mechanisms.
a. Gene amplification: This occurs when a constant stimulus exists for a long time, e.g. dihydro­
folate reductase gene gets amplified (increase in the number of DHFR genes) in a person on
antifolate drugs (for cancer).
b. Gene switching: When a person is exposed to a foreign antigen for the first time, immu­
noglobulin M (IgM) is produced. Later, by gene switching for the same antigen, IgG is
produced.
c. Rearrangement of genes in DNA: Depending on the nature of antigens exposed, many types
of antibodies can be produced from a limited number of J, V and C segments. This is by
rearrangement of genes.
d. Control of transcription: This is done by cis and trans acting elements on DNA like enhanc­
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ers/silencers, hormones binding to hormone response elements, various proteins binding
to DNA, histone modifications, etc.
e. Post-transcriptional regulation: Post transcriptional modifications like 5’ capping, poly A tail,
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splicing, etc. can regulate the stability of RNA. A transcribed monocistronic RNA can form
different proteins by RNA editing (alteration of a base in mRNA), e.g. apolipoproteins Apo
B100 and Apo B48 are formed from the same RNA.
f. Translational regulation: This is not a major mechanism in eukaryotes. For example, heme
can block its own synthesis by inhibiting the production of enzymes responsible for it.

Key Point
Cistron: Smallest unit of gene expression. It codes for one peptide or a subunit of protein.

Ch-16.indd 214 21-06-2014 11:52:07
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DNA Replication   | 

Contd...
Reverse Transcriptase
• Temin and Baltimore isolated this enzyme in 1970 DNA Repair
• They are RNA Dependent DNA Polymerase damaging Defects in mecha- Disorder associ-
• Synthesize new DNA strand with RNA as template agents DNA nism ated
• Thus, they reverse Central dogma of molecular genetics Werner Syndrome
• These enzymes are important in RNA Viruses like Retroviruses (WS)
• Telomerase has reverse transcriptase activity. Rothmund-Thomson
m

Syndrome (RTS)
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DNA REPAIR MECHANISMS Breast Cancer Sus-

ceptibility (BRCA 1,
• DNA is subjected to a huge array of chemical, BRCA 2)
physical, and biological assaults on a daily basis 1. UV lightQ 1. Bulky Ad- Nucleotide Xeroderma Pigmen-
• Repair of damaged DNA is critical for maintaining 2. Chemi- ducts Excision tosaQ (XP)
cals 2. Pyrimidine Repair Cockayne Syn-
genomic integrity and thereby preventing the
DimersQ (NER)Q drome (SS)
propagation of mutations
Trichothiodystrophy
• Eukaryotic cells contain five major DNA repair (TTD)
pathways.
1. Oxygen 1. Abasic Sites Base MUTYH–associated
The mechanisms of DNA repair include: radicals 2. Single Excision Polyposis (MAP)
2. Alkyl- strand Repair
• Nucleotide Excision Repair (NER)
ating breaks (BER)
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• Mismatch Repair (MMR) agents 3. 8 oxogua-

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• Base Excision Repair (BER) nine lesions

• Homologous Recombination (HR) 1. Replica- 1. Bases mis- Mismatch Hereditary non-
• Nonhomologous End-Joining (NHEJ) repair. tion er- match repair polyposis
rors 2. Insertion (MMR) Colorectal Cancer
DNA Repair (HNPCC)Q (Lynch
damaging Defects in mecha- Disorder associ- 3. Deletion
syndrome)
agents DNA nism ated
1. Ionizing 1. Double Nonhomol- Severe Combined
Radia- Strand ogous End Immunodeficiency Double Strand Break Repair Mechanisms (DSB)
tions BreaksQ Joining (SCID) • They are Homologous recombination (HR) and non
2. X-raysQ 2. Single (NHEJ)
Strand
Homologous End Joining Repair (NHEJ)
3. Anti-
tumor Breaks Nonhomologous end joining
drugs 3. Intrastrand Homolo- Ataxia Telangecta- Homologous recombination repair
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cross links gous Re- sia Like Disorder Major mechanism of DSB repair Major mechanism of DSB repair
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4. Interstrand combina- (ATLD) in yeast mammals

cross links tion (HR) Nijimen break Syn-
drome (NBS) Takes place between homolo- Does not need a homologous
Bloom’s Syndrome gous chromosomes Chromosome
(BS) Takes place before cell enter Takes place before cell enter
mitosis (S and G2/M phase) mitosis (G0/G1 phase)
Contd...

REVIEW QUESTIONS

DNA REPLICATION c. DNA replication proceeds in one direction

d. Lagging strand stick by RNA primase
1. False statements is/are:
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a. In leading strands DNA is synthesized e. DNA polymerase III-processive leading strand

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continuously synthesis
b. Multiple origins of replication are possible for Ans. b, c, d. (Harper 30/e page 381-387)
bacteria • Multiple ori in eukaryotes
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• DNA replication is bidirectional 3. Incorrect statements are:

• Lagging strand stick by DNA Ligase a. T4 DNA polymerase has 3’->5’ exonuclease
activity
DNA Replication
b. Klenow fragment of DNA polymerase I function
Salient Features of DNA Replication is almost similar to T4 DNA polymerase
• Occurs in the S Phase of the cell cycle c. Restriction endonuclease cut DNA chains at
m

• Each DNA Stand separate and each acts as template specific location
strand on which complementary strand is synthesized
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d. Endonuclease cut DNA at 5’ terminus

• Base pairing rule is obeyed e. Right-handed helix of DNA is more common
• Semi conservative nature-Proved by Meselson and Ans. d. Endonuclease cut DNA at 5’ terminus.
Stahl Experiment Endonuclease cut the DNA from within
• New Strand is synthesized in the 5’ to 3’ direction Exonuclease cuts the DNA from ends
• Synthesis of DNA in both strands are not similar
T4 DNA Polymerase similar to Klenow polymerase.
‒ Leading strand: The strand which DNA is
continuously polymerized 4. Which DNA polymerase is involved in repair of
mammalian DNA:
‒ Lagging Strand: The strand which is DNA is dis-
a. Alpha
continuously polymerized (Semi discontinuous)
b. Beta
• Replication proceeds from multiple origins in each
c. Gamma
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chromosome in eukaryotes including humans (a total

d. Epsilon
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of as many as 100 in humans)

• Replication obeys polarity e. Delta
• Replication occurs in both directions along all of Ans. b. Beta (Ref: Harper 30/e page 383)
the chromosomes, i.e. bidirectional in prokaryotes Eukaryotic DNA Polymerase
and eukaryotes Mainly five types of Eukaryotic DNA Polymerase
• Both strands are replicated simultaneously • DNAP α
• Replication process generates ‘replication bubbles’. • DNAP β
• DNAP γ
• DNAP δ
• DNAP ε
Eukaryotic DNA polymerase Function
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DNAP alpha Primase

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DNAP beta DNA repairQ

DNAP gamma Mitochondrial DNA synthesis
Fig. 10.2: Direction of replication DNAP delta Lagging strand synthesis
DNAP epsilon Leading strand synthesis
2. True about Eukaryotic DNA replication compared
to prokaryotic: Prokaryotic DNA Polymerase
a. Conservative Three types of Prokaryotic DNA Polymerase
b. Semi conservative • Pol I
c. Unidirectional • Pol II
d. Bidirectional • Pol III
e. Semi discontinuous Prokaryotic DNA
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Ans. b, d, e. Semi conservative, Bidirectional, Semi Polymerase Function

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discontinuous. (Harper 30/e page 381-387) Pol I Gap filling following DNA replication, repair,
and recombination
This questions means the common features between
eukaryotic and prokaryotic DNA replication. Contd...
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DNA Replication   | 

Contd... • This results in shortening of DNA with each cell

Prokaryotic DNA division
Polymerase Function • This is prevented by presence of Telomere and
Pol II DNA proofreading and repair Telomerase.
Pol III Processive, leading strand synthesis, Telomeres
Okazaki fragment synthesis
• Are tandem repeats of simple sequence on 3’ end of
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5. The gaps between segment of DNA on the the parent DNA

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lagging stand produced by restriction enzymes • In humans repeats are (TTAGG)n.

are joined/sealed by: (AI 2009) Telomerase (Telomere Terminal Transferase)
a. DNA Ligases • Enzyme which prevents shortening of DNA
b. DNA Helicase • Has an intrinsic RNA primer
c. DNA Topoisomerase • Has Reverse Transcriptase (RNA Dependent DNA
d. DNA Phosphorylase Polymerase) activity
Ans. a. DNA Ligase (Ref: Harper 30/e page 381) • Present in Germ line, stem cells, most cancer cells
• Absent from most somatic cells.
6. During replication of DNA, which one of the
following enzymes polymerizes the Okazaki Clinical Significance of Telomerase
fragments? • Absence of Telomerase lead to premature aging
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a. DNA Polymerase I • In Cancer cells increased Telomerase activity

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b. DNA Polymerase II • Telomerase has become an attractive target for cancer

c. DNA Polymerase III chemotherapy and drug development.
d. RNA Polymerase I
8. DNA Polymerase with both replication and repair
Ans. c. DNA Polymerase III (Harper 30/e page 381-387) function is:
DNA Synthesis a. I
On 5’----> 3’ Strand (Lagging Strand) (Discontinuous b. II
Strand) (Retrograde Strand) c. III
Synthesized in discontinuous manner d. None of the above
Small fragments of DNA are in short spurts of 100–250 Ans. a. I and b. II (Harper 30/e page 383)
nucleotide (1000–2000 bp in prokaryotes) called Okazaki 9. Radiolabelled DNA was allowed to replicate
Fragment synthesized in 5’ --- 3’ direction.
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twice in a nonradioactive environment. Which of

• By DNAP III in prokaryotes.
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the following is true?

• By DNAP δ in eukaryotes. a. All the strands will have radioactivity
b. Half of the DNA will have no radioactivity
7. All of the following cell types contain the enzyme
telomerase which protects the length of telome- c. No strands will have radioactivity
rase at the end of chromosomes, except: d. Three–fourth of the DNA replicated will have
a. Germinal radioactivity.
b. Somatic Ans. b. Half of the DNA will have no radioactivity
c. Hemopoietic (Harper 30/e page 381-387)
d. Tumor • Semi conservative nature of DNA Replication proved
by Meselson and Stahl states that half of the parent
Ans. b. Somatic (Harper 30/e page 374)
strand is conserved during replication in the daughter
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Telomere and Telomerase strand

• On the 3’ end of the linear DNA, RNA primer is
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• After one replication all the DNA will have radio-

removed by RNAaseH, which leaves a gap at the activity
3’ end • After two replication half of the DNA will have
• Thus 5’ end of the new strand is not synthesized radioactivity.
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10. In which of the following phase, DNA doubling 13. Action of telomerase is:
occurs: a. DNA repair
a. Gl phase b. Longevity of cell
b. S phase c. Breakdown of telomere
c. G2 phase d. None
d. M phase Ans. b. Longevity of cell-aging (Harper 30/e page 374)
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Ans. b. S Phase (Harper 30/e page 381-387)

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14. Ends of chromosomes replicated by:

11. Unwinding Enzyme in DNA synthesis: a. Telomerase
a. Helicase b. Centromere
b. Primase c. Restriction endonuclease
c. DNA Polymerase d. Exonuclease
d. Transcriptase Ans. a. Telomerase (Harper 30/e page 374)
Ans. a. Helicase (Ref: Harper 30/e page 382)
15. Highly repetitive DNA is seen in:
Classes of Proteins involved in DNA Replication
a. Cloning of DNA
Protein Function
b. Microsatellite DNA
DNA polymerases Deoxynucleotide polymerization
c. Telomere
Helicases Processive unwinding of DNA
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d. Centromere
Topoisomerases Relieve torsional strain that results from
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helicase-induced unwinding Ans. c, d. Telomere and Centromere

DNA primase Initiates synthesis of RNA primers (Harper 30/e page 377)
Single-strand binding Prevent premature reannealing of dsDNA • In human DNA, at least 30% of genome consist of
proteins repetitive sequence
DNA ligase Seals the single strand nick between the • These sequences are clustered in the centromere and
nascent chain and Okazaki fragments on
and telomere
lagging strand
• They are transcriptionally inactive
• They are mostly having structural role in the
12. True about telomerase or telomere is/are:
chromosome.
a. They are present at the ends of eukaryotic 16. Which enzymatic mutation is responsible for
chromosome immortality of cancer cells:
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b. Increased telomerase activity favors cancer a. DNA reverse transcriptase

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cells b. RNA polymerase

c. DNA dependent RNA polymerase c. Telomerase
d. DNA polymerase d. DNA polymerase
Ans. a, b. They are present at the ends of …, increased Ans. c. Telomerase (Robbins 9/e page 288)
telomerase… (Harper 30/e page 374) Telomerase is one of several factors that contribute to
• Telomeres are present in the ends of eukaryotic the endless replicative capacity (the immortalization) of
chromosomes. cancer cells.
• Telomeres consist of TG repeats.
• Telomere shortening has been associated with 17. Okazaki fragments are formed during the
malignant transformation and aging synthesis of:
• Telomerase, multisubunit RNA template containing a. dsDNA
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RNA Dependent DNA Polymerase (Reverse b. ssDNA

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Transcriptases). c. mRNA
• Enzyme responsible for Telomere synthesis and d. tRNA
maintaining the length of telomere. Ans. a. ds DNA
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DNA Replication   | 

18. Correct sequence of enzymes required for DNA DNA Repair

formation is: 21. Xeroderma pigmentosa is due to:
a. DNA polymerase → protein unwinding a. Base excision defect
enzyme → DNA ligase → DNA Isomerase → b. Nucleotide excision defect
Polymerase I
c. SOS repair defect
b. Protein unwinding enzyme → polymerase
d. Cross linking defect
I → DNA ligase → DNA isomerase → DNA
m

polymerase Ans. b. Nucleotide excision defect (Harper 30/e page 389)

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c. RNA polymerase → DNA polymerase III → DNA damag- Defects in Repair Disorder
DNA polymerase I → DNA ligase ing agents DNA Mechanism associated
d. RNA polymerase → DNA polymerase III • Ionizing Ra- • Double N o n h o m o l o - Severe Com-
→ DNA ligase → exonuclease → DNA diations Strand gous End Join- b i n e d I m m u-
polymerase I • X-rays Breaks ing (NHEJ) nodeficiency
(SCID)
Ans. c. RNA Polymerase → DNA polymerase III → DNA • Antitumor • Single Strand
Breaks
polymerase I → DNA ligase (Harper 30/e page 381) drugs
Homologous Ataxia Tel-
• Intrastrand
• The correct sequence of enzymes is Helicase, Primase, Recombination angiectasia
cross links
DNA Polymerase III, DNA Polymerase I and on (HR) Like Disorder
• Interstrand Nijmen Break
lagging strand, DNA ligase cross links Syndrome
• Helicase, Primase, DNA Polymerase III on leading Bloom’s Syn-
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strand. drome, Werner

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Syndrome,
19. True about DNA polymerase in eukaryotes: Rothmund-
Thomson
Syndrome
a. Components are α, β, γ, δ, ε
Breast Cancer
b. β associated with repair Susceptibil-
c. γ associated with repair ity (BRCA 1,
BRCA 2)
d. δ associated with synthesis of mitochondria
DNA • UV light • Bulky Ad- Nucleotide Ex- Xeroderma
ducts cision Repair Pigmentosa
e. α is abundant amount • Chemicals
(NER) Cockayne Syn-
• Pyrimidine
Ans. a, b. Components are ..., β associated with ... Dimers
drome
Trichothio-
(Harper 30/e page 381)
dystrophy
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Eukaryotic DNA polymerase Function

• Oxygen radi- • Abasic Sites Base Excision MUTYH –
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DNAP alpha Primase cals Repair (BER) associated

• Single strand
DNAP beta DNA repair Polyposis
• Alkylating breaks
DNAP gamma Mitochondrial DNA synthesis agents • 8 oxoguanine
DNAP delta Lagging Strand Synthesis lesions
DNAP epsilon Leading Strand Synthesis • Replication • Bases mis- Mismatch Hereditary
errors match repair (MMR) nonpolyposis
20. DNA polymerase have: • Insertion Colorectal
Cancer
a. 3’–5’ polymerase activity • Deletion (HNPCC)
b. 5’–3’ polymerase activity
c. 3’–5’ exonuclease activity
22. UV light damage to the DNA leads to:
d. 5’–3’ exonuclease activity
Ans. b, c, d. 5’–3’ polymerase activity, 3’–5’ exonuclease
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a. Formation of pyrimidine dimers

activity, 5’–3’ exonuclease activity Harper 30/e page 382
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b. No damage to DNA
DNA Polymerase have 5’ to 3’ Polymerase activity, 3’–5’
exonuclease (Proofreading) and 5’–3’exonuclease activity c. DNA hydrolysis
(repair) activity. d. Double stranded breaks
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Ans. a. Formation of pyrimidine dimers • This is repaired by Nucleotide excision repair (NER)
(Harper 30/e page 390) • Defect in NER leads to Xeroderma Pigmentosa.
DNA lesions formed by UV light damage are Bulky 25. Which of the following is true regarding DNA
adducts and Pyrimidine Dimers. double-strand breaks repair pathway:
a. Homologous recombination require a long
23. Excessive ultraviolet (UV) radiation is harmful to
homologous sequence to guide repair
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b. Nonhomologous end-joining does not require
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by ultraviolet radiation I by:

a. Inhibition of DNA synthesis a long homologous sequence to guide repair
b. Formation of thymidine dimers c. Homologous recombination repairs DNA
before the cell enters mitosis
c. Ionization
d. Nonhomologous end-joining repairs DNA
d. DNA fragmentation
before the cell enters mitosis
Ans. b. Formation of thymidine dimers e. Nonhomologous end-joining is prominent
(Harper 30/e page 390) DSB repair mechanism in mammals.
24. The primary defect in Xeroderma pigmentosa is: Ans. a, b, c, d, e.
Double Strand Break Repair Mechanisms (DSB). They are
a. Formation of thymidine dimers Homologous recombination (HR) and Nonhomologous
End Joining Repair (NHEJ).
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c. Exonuclease is defective Nonhomologous end joining

d. Formation of adenine dimers Homologous recombination repair
Major mechanism of DSB repair Major mechanism of DSB repair
Ans. a. Formation of Thymidine dimers
in yeast mammals
(Harper 30/e page 390) Takes place between homolo- Does not need a homologous
• UV light radiation causes Bulky adducts and gous chromosomes Chromosome
Pyrimidine dimers (Most common is Thymidine Takes place before cell enter Takes place before cell enter
dimers) mitosis (S & G2/M phase) mitosis (G0/G1 phase)
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