DNA replication by Dr. YALAVARTHI NAGARAJU.pptx

DNA
replication
By
Dr. YALAVARTHI NAGARAJU

List of Contents
1. Purpose
2. A brief History of DNA
3. Review on DNA structure
4.

• Cells need to make a copy of DNA before
dividing so each daughter cell has a complete
copy of genetic information
Chromosomes form during cell division
Duplicate to yield a full set in daughter cell
Purpose

Brief history of DNA
• Friedrich Miescher (1844-1895) first time sep. nuclei and ext. nuclein (P-rich: NA+Proteins) from
cytoplasm
• Levine (1869-1940), NAs can be broken into smaller sections of nucleotides.
• Early scientists thought protein was the cell’s hereditary material because it was more complex than
DNA
• Proteins were composed of 20 different amino acids in long polypeptide chains
• Fred Griffith worked with virulent S and nonvirulent R strain Pneumoccocus bacteria
• He found that R strain could become virulent when it took in DNA from heat-killed S strain
• Study suggested that DNA was probably the genetic material
• Transformation is a special type of recombination in which a segment from the transforming
DNA replaces the homologous segment of the bacterial chromosome.

Radioactive 32P was injected into bacteria!
• Avery, Mac Leod & Mc Carty (1944) carried the experiments of Griffith invitro
Found that DNA is the transforming Principle
• Bacterial Conjugation: Laderberg & Tatum (1946)
Unidirectional transfer of F+ to F- and later converted to partial diploid in E. coli
• Chromosomes are made of both DNA and protein
• Experiments on bacteriophage viruses by Hershey & Chase proved that DNA was
the cell’s genetic material

Discovery of DNA Structure
• Erwin Chargraff showed the amounts of the four bases on DNA
(A,T,C,G)
• In a body or somatic cell:
A = 30.3%
T = 30.3%
G = 19.5%
C = 19.9%

Chargaff’s(1940’s) Rule
• All nucleotide bases were not present in equal amounts.
• The ratio of the diff bases changed between diff species.
• The no. of Pyrimidine bases (C+T) is equal to no. of Purine bases (A+G).
• There is an Law of Equivalence between the bases with amino groups at 4 or 6 position
(A+C) and bases with keto groups at 2&4 position (T+G).
• Adenine must pair with Thymine
• Guanine must pair with Cytosine
• The bases form weak hydrogen bonds
T A G C

DNA Structure
• Rosalind Franklin, Wilkins & co took diffraction x-ray
photographs of DNA crystals- Multistrand with a dia of 22Å and
groups spaced at an interval of 3.4 Å along the fibre &
occurrence of a repeating unit every 34 Å.
• Franklin had discovered that DNA could crystallize into two
different forms, an A form and a B form.
In the 1953, Watson & Crick built the first model of DNA
using Franklin’s x-rays crystallography & chemical
analysis- proposed Double Helix Model of DNA

Little review on DNA structure
DNA is made up of subunits called NUCLEOTIDES
 Each nucleotide is made up of 3 basic parts:
* 5-carbon sugar: deoxyribose
* nitrogenous base: A, G, C, or T
* 1 phosphate group

Watson and Crick Calculations based on Franklin’s work
a) DNA is a helix with a width of 2 nm
b) purine & pyrimidine bases are stacked 0.34 nm apart
c) the helix makes 1 full turn every 3.4 nm along its length
d) there are 10 layers of bases (or rungs) in each turn of the helix
e) to be consistent with a 2 nm width, a purine on one strand must
pair (by H-bonding) with a pyrimidine on the other strand
f) base structure dictates which pairs of bases can form hydrogen
bonds

The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G
with C.
The first step in replication
is separation of the two
DNA strands.
Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
“The Beauty of the model was that the structure of DNA suggested
the basic mechanism of its replication”

Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data

DNA replication facts
• DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to
copy their DNA.
• In the process of "replication" each strand of the original double-stranded DNA molecule serves as template for
the reproduction of the complementary strand.
• Two identical DNA molecules have been produced from a single double-stranded DNA molecule.
• DNA has to be copied before a cell divides
• DNA is copied during the S or synthesis phase of interphase
• New cells will need identical DNA strands
• Each cell division cell must copy its entire DNA
• So each daughter cell gets a complete copy
• Rate of synthesis
• Bacteria = 1000 bases per second
• Mammals = 100 bases per second
• Problem - with a single replication origin in DNA
• Bacteria genome is 4 x 106. Takes 20 minutes to copy.
• Human is 3.2 x 109. Would take 10,000 times longer.
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
interphase
DNA replication takes
place in the S phase.
S
phase

Semi-Conservative Model
• Replication of DNA
• The 2 DNA strands separate (hydrogen bonds break;
unwind; unzip DNA strand)
• Base pairing allows each strand to serve as a template for
a new strand
• New strand is 1/2 parent template &
1/2 new DNA
• Nucleotides line up singly along the template strand (A-T,
G-C) (new hydrogen bonds form)
• Enzymes link the nucleotides together at their sugar-
phosphate groups (phosphodiester bonds)

1) Semiconservative Replication:
DNA Replication would create two molecules. Each of them would be a complex of an old (parental and a daughter strand).
2) Conservative Replication:
DNA Replication process would create a brand new DNA double helix made of two daughter strands while the parental chains would
stay together.
3) Dispersive Replication:
Replication Process would create two DNA double-chains, each of them with parts of both parent and daughter molecules.
As we will see at the next chapter, the correct model is the first. Semiconservative DNA Replication was proved by the experiment of
Meselson - Stahl.

DNA replication by Dr. YALAVARTHI NAGARAJU.pptx

DNA Replication
• The entire chromosome must be replicated precisely once for every cell
division
• The unit of DNA replication is referred to as a replicon
• The genome of a prokaryote constitutes a single replicon
• Any DNA molecule that contains an origin can replicate autonomously in
the cell
• Three steps are involved in replication:
i. Initiation
ii. Elongation
iii. Termination

Prerequisites for DNA replication
DNA replication requires:
1. A DNA template
2. DNA Gyrase
3. DNA helicase (Dna B)
4. The DnaA and Dna C protein
5. Single-strand DNA Binding Proteins (SSB)
6. Primase (Dna G)
7. DNA Pol
8. Ligase

Initiation
1st step: unwinding of DNA
• It involves the recognition of an origin of replication by a complex of proteins
• Before DNA synthesis the parental strands must be separated and stabilized in the single
stranded form
• The synthesis of the daughter strands can be initiated at the replication fork (Y-shaped region),
New strands grow at the forks
• The act of initiating synthesis of DNA strand is accompanied by a protein complex called the
primosome in E.coli
• DNA helicase: unwinds the part of DNA helix by breaking weak hydrogen bonds
• Single-Strand Binding Proteins attach and keep the 2 DNA strands separated and untwisted
• DNA gyrase (Topoisomerase) attaches to the 2 forks of the bubble to relieve stress on the DNA
molecule as it separates
• As the 2 DNA strands open at the origin, Replication Bubbles form
• Eukaryotic chromosomes have MANY bubbles
• Prokaryotes (bacteria) have a single bubble

• As the ds DNA opens up DNA is synthesized only in
the 5’-3’ direction and never in the 3’-5’ direction for the
complementary strand
• In the 3’-5’ template strand the DNA synthesis made
in the 5’-3’ direction in a continuous way is called the
leading strand
• On the other template short pieces of DNA ( ~1000
nucleotides long) in the 5’- 3’ direction are made and
the pieces are joined together
• The leading and lagging strands are synthesized
simultaneously by a single dimeric DNA polymerase III
complex

• Dna G primase associates transiently with the primosome and is activated by
Dna B to initiate the synthesis of the primer which is 11 –12 bases long
• It synthesizes primers starting with the sequence pppCAG, opposite the
sequence 3’-GTC in the template
• The primosome moves in the anti elongation direction along with the parallel
strand from 5’ toward 3’ direction
• The primosome moves in the same direction of the replication fork but in the
opposite direction from Dna synthesis of the lagging strand
• Dna B has a helicase action that generates the replication fork in DNA
• It moves in the 5’ –3’ direction

2nd step: RNA primase
• RNA primase: adds a small section of RNA (RNA primer) to the 3’ end of template DNA
• RNA primers are used to initiate a new strand

3rd step: building daughter DNA strands
• DNA polymerase III (enzyme that builds new DNA strand) can only add nucleotides to
existing strands of DNA
• DNA replication is continuous on one strand and discontinuous on the other strand
• Synthesis of a new strand on DNA template is always in the 5’ 3' direction
• The 5' triphosphate can only be added to a free 3'OH of deoxyribose.
• The two antiparallel strands are replicated simultaneously in both directions.
• The parent strand at the 3' end of the template determines the daughter or leading strand
in continuous replication
• The parent strand at the 5' end of the template produces the lagging strand as short
pieces of DNA (100-200 nucleotides in eukaryotes and longer in prokaryotes)

Elongation
• Elongation is undertaken by another complex of proteins. The RNA primer is extended by
DNA Pol III
• The replisome is the unit involved in elongation which does not exist physically as an
independent unit but it is assembled from its components at the site of replication
• As the replisome moves along DNA , the parental strands unwind and the daughter
strands are synthesized
• This causes the NEW strand to be built in a 5’ to 3’ direction
RNA
Primer
DNA Polymerase
Nucleotide
5’
5’ 3’

• DNA Pol synthesizes DNA for both the leading and lagging strands
• After DNA synthesis by DNA Pol III DNA pol I uses its 5’-3’ exonuclease activity to
remove the primer and then fills the gaps with a new 5’ – 3’ exonuclease activity
• Finally, DNA pieces are joined together by the DNA ligase
• The entire DNA-synthesizing complex at each replication fork, which also includes
topoisomerase, helicase, and primase, is sometimes referred to as a replisome

Remember HOW the Carbons Are Numbered!
CH2
O
C1
C4
C3 C2
5
Sugar
(deoxyribose)
O
O=P-O
O
Phosphate
Group
N
Nitrogenous base
(A, G, C, or T)

P
P
P
O
O
O
1
2
3
4
5
5
3
3
5
G C
T A
P
P
P
O
O
O
1
2 3
4
5
5
3
5
3

• The Leading Strand is synthesized as a single strand from the
point of origin toward the opening replication fork
RNA
Primer
DNA Polymerase
Nucleotides
3’
5’
5’

• The Lagging Strand is synthesized discontinuously against overall
direction of replication
• This strand is made in MANY short segments It is replicated from the
replication fork toward the origin
RNA Primer
Leading Strand
DNA Polymerase
5’
5’
3’
3’
Lagging Strand
5’
5’
3’
3’

• Okazaki Fragments - series of short segments on the lagging strand
• Must be joined together by an enzyme
Lagging Strand
RNA
Primer
DNA
Polymerase
3’
3’
5’
5’

• The enzyme Ligase joins the Okazaki fragments together to make one strand
Lagging Strand
Okazaki Fragment 2
DNA ligase
Okazaki Fragment 1
5’
5’
3’
3’

Removal of RNA primer
• DNA polymerase III dissociates, leaving a "nick" (a single stranded gap) between the new
DNA and the primer
• After the last ribonucleotide is removed from the primer and replaced with a
deoxynucleotide, there is still a nick in the newly synthesized lagging strand
• This nick is closed by DNA ligase, which forms a covalent phosphodiester bond between
the Okazaki fragments, joining them into a continuous strand of DNA
• Starting at the nick, DNA polymerase I removes the primer RNA using its 5' to 3'
exonuclease activity, and replaces them with dNTPs, using its DNA polymerase activity

Termination
• At the end of replication joining and /or termination are necessary
• The separation of the circular ds DNA is carried out with topoisomerases
• Replacement of RNA primer by DNA polymerase I
• The sequence that stop the movement of replication fork are identified as ‘ter’ elements
• These are 23 bp consensus sequences that provide the binding site for the ‘tus’ gene, a
36 kD Protein needed for the termination
• Replication forks meet at Ter sequences (t locus)
• Ter (termination) sequences 20 bp long
• core consensus is : 5’-GTGTGTTGT-3’
• bound by Tus protein (terminus utilization substance)
• not essential for termination of replication
TerG TerF TerB TerC TerA TerD TerB
clockwise fork trap
counterclockwise
fork trap
Clockwise
fork
Counterclockwise
fork
oriC

Catenanes
• Catenanes
- interlinked circular chromosomes
- separation requires E.coli DNA topoisomerase IV
(type II topoisomerase)

Proofreading New DNA
• DNA polymerase initially makes about 1 in 10,000 base pairing errors
• Enzymes proofread and correct these mistakes
• The new error rate for DNA that has been proofread is 1 in 1 billion base pairing errors

DNA Damage & Repair
• Chemicals & ultraviolet radiation damage the DNA in our body cells
• Cells must continuously repair DAMAGED DNA
• Excision repair occurs when any of over 50 repair enzymes remove damaged parts of
DNA
• DNA polymerase and DNA ligase replace and bond the new nucleotides together

Repliosome complex
• The entire DNA-synthesizing complex at each replication fork, which also
includes topoisomerase, helicase, and primase, is sometimes referred to
as a replisome

General Mode of DNA Replication
• The DNA molecule is unwound and prepared for synthesis by the action of DNA
gyrase (supercoiling), DNA helicase (double helical structure) and SSB
• Replication forks: When the two strands of double helical DNA separate and
replication of both strands begins, a forked or Y-shaped structure is formed
Bi-directional replication:
• In bacterial cells, replication starts at a specific origin of replication within the
circular DNA molecule and proceeds in both directions away from the origin
• This results in the formation of a replication "bubble", which continues to
elongate as replication proceeds

• A free 3’-OH group is required for replication
• There are no enzymes capable of initiating the synthesis of a DNA-templated DNA molecule at
the level of a single nucleotide which makes it necessary to use an indirect priming procedure
• New DNA synthesis is primed with a short segment of RNA that is later removed
• A separate enzyme in the initiation complex called primase synthesizes a short RNA primer
each time that new DNA synthesis begins, including all new starts in the discontinuous pattern
of synthesis
• DNA replication begins at a specific site (origin of replication) characterized by the presence of
repeated 9 base and 13 base nucleotide sequences
• The consensus units are referred to as 9-mer (Dna A box) and 13-mer
• The 13 mers are AT rich, making easier to separate the two strands of the double-stranded
DNA.

Bi-directional replication
• In circular bacterial chromosome replication starts at the origin of replication (ori C)
• Replication is bi-directional
• Two forks move in opposite directions and meet eventually at a locus
• This results in the formation of a replication "bubble", which continues to elongate as
replication proceeds
• The region of replicating DNA associated with the single origin is called replication bubble or
eye (θ ) and consists of two replication forks moving in opposite directions
• The double helix opens up and both strands serves templates of the synthesis of DNA
• The primosome thus consists of 6 proteins, Pri A, Pri B, Pri C, Dna T , Dna B, and Dna
C
• Dna B is the central component that provides the 5’ – 3’ helicase activity
• A free 3’-OH group is required for replication
• There are no enzymes capable of initiating the synthesis of a DNA-templated DNA molecule at
the level of a single nucleotide which makes it necessary to use an indirect priming
procedure
• New DNA synthesis is primed with a short segment of RNA that is later removed

Unidirectional synthesis of antiparallel DNA
• A separate enzyme in the initiation complex called primase synthesizes a short
RNA primer each time that new DNA synthesis begins, including all new starts in
the discontinuous pattern of synthesis
• At any replication fork, one of the template strands has a 3' to 5' orientation,
which is needed for the synthesis of a new complementary strand in a 5' to 3'
direction
• The other template strand has a 5' to 3' orientation and is thus unable to support
synthesis beginning at the origin and moving away from it in a 3' to 5' direction
• The strand whose synthesis begins immediately is called the "leading" strand,
and the one whose synthesis is delayed is called the "lagging" strand.

Question:
• What would be the complementary DNA strand for the following DNA sequence?
DNA 5’-CGTATG-3’

Answer:
DNA 5’-GCGTATG-3’
DNA 3’-CGCATAC-5’

Enzymes involved in DNA synthesis

oriC (origin of replication)
• OriC is a region of DNA approximately 240 nucleotides long
• It contains repetitive 9-base pair and 13-base pair sequences (known as the '9-mer' and
'13-mer' regions).
• These sequences are AT rich regions, which melt at lower temperatures than DNA
containing GC pairs.
• These regions are postulated to help melt the DNA duplex in the oriC region for initiation
of DNA replication.

• Topoisomerases are enzymes that convert one
topological version of DNA into another
• They do it by changing the linking number
Topoisomerase I
• Topoisomerases I change the linking number in steps
of 1.
• They pass a single DNA strand through a nick
Topoisomerase II
• Topoisomerases II change the linking number in
steps of 2 by passing both strands of double-
stranded DNA through a break.
Proteins involved in replication

DNA gyrase
• The enzyme Gyrase catalyzes the formation of –ve supercoiled coils that help in the
unwinding process
• Separation of the two strands of the DNA double helix requires substantial unwinding of
the helix
• As replication proceeds in both directions around the circular chromosomes of bacteria, a
structure reminiscent of the Greek letter theta (θ ) is formed
• Replication continues until the replication bubbles fuse to yield fully replicated DNA
strands

DNA Helicase (Dna B protein)
• Helicase accomplishes unwinding of the original double
strand, once supercoiling has been eliminated by the
topoisomerase
• There are a number (~10) proteins in E.coli that have
helicase activity
• These belong to the class of Topoisomerases II
•(DnaB protein) helicase is a hexamer and requires the energy of ATP
hydrolysis to unwind the helix
•dnaB protein binds to the single stranded DNA in the general region of the
oriC DNA segment.
•Binding requires ATP as well as the dnaC gene product (the dnaC protein).
•After helicase/dnaC binds to the DNA, the dnaC protein is released.
•Two helicases bind at the oriC region, one helicase on each strand of the
DNA.

Dna A protein
• The DnaA protein initiates DNA replication, creating new replication forks
• The Dna A protein binds at the ori C sequence, and is a regulator of the frequency of initiation
of replication in E. coli.
• The protein coded by the DnaA gene binds to the repeated 9 mer
• This forms a tight loop and generates a strain that causes strand separation in the region
containing the AT-rich 13-mer
• Strains of E. coli with mutations in the dnaA gene were able to grow at 30 °C, but not at 39-42
°C.
• However, if DNA synthesis was begun at 30 °C, and then the temperature was shifted to 42
°C, DNA synthesis continued until the genome was replicated (and the cell divided), but no
new initiation of DNA synthesis was possible.
Conclusion: Somehow the product of the dnaA gene (i.e. the dnaA protein) is required
for initiation of DNA synthesis.

DnaA protein (conti…)
• dnaA protein binds to the '9-mer' region in oriC and forming a multimeric complex
with 10-20 protein subunits (i.e. at a single oriC region there will be bound 10-20
dnaA protein molecules).
• Binding requires ATP.
• Further addition of ATP was observed to result in a melting and opening up of the
DNA duplex in the oriC region. This was determined by addition of S1
nuclease (like mung bean, but will also cut DNA at the site of an internal nick),
which resulted in cleavage of DNA at the site of oriC.

Single-strand DNA Binding Proteins (SSB)
• Single stranded DNA-binding proteins (SSBPs) attach to the single stranded DNA
generated by unwinding the double helix and temporarily keep it from reforming double
helical structures
• These proteins bind single-stranded DNA at the replication fork and physically blocks
potential hybridization
• The single stranded DNA is kept away from coiling and protected from the action of
nucleases by SSBs
• These proteins bind to DNA as a tetramer and stabilize the single stranded structure
• Replication is ~100 times faster when these proteins are attached to the ssDNA

DNA Polymerases
• DNA Polymerase I was the first enzyme to be discovered A. Kornberg in 1955 with polymerase activity.
It is chiefly a DNA repair enzyme and is used for invitro DNA replication.
• DNA pol (holoenzyme) is dimeric in nature
• Bacteria have 3 types
• DNA Pol I, II, and III
• DNA Pol III involved in replication of DNA
• DNA Pol I involved in repair
• Humans have 4 types (you need to know, now)
• DNA Pol alpha, beta, delta - nuclear DNA
• DNA Pol gamma - mitochondrial DNA
• Three activities are associated with it
i. 5’ – 3’ elongation (Polymerase act)- primer extension
ii. 3’ –5’ exonuclease activity (Proof reading act)- one nt is removed at a time. Proof reading reduces the
errors by 100 fold.
iii. 5’ –3’ exonuclease activity (repair act)- Excision of DNA during repair. It removes 10 bases at a time. It
digest NA from one end and not make internal cuts

Theodor Hanekamp © 2003 5
DNA polymerases in E. coli
• DNA polymerase I
– involved in clean-up functions, DNA repair, etc.
• DNA polymerase II
– involved in DNA repair
• DNA polymerase III
– principle replication enzyme in E. coli
– fast (250 –1000 nts/sec) and high processivity
• DNA polymerase IV and V
– discovered in 1999
– involved in specific forms of DNA repair

DNA pol I
• DNA Pol I is a template directed enzyme
• DNA Pol I is a monomeric protein with three active sites
• It contains both the polymerizing and the proof reading activity
• It adds complementary nucleotides to the free 3’-OH group of the primer
• DNA pol 1 Is encoded by gene polA, has a single polypeptide (monomeric) and can initiate
replication in vitro at a nick in a DNA duplex.
• DNA Pol I when cleaved with subtilisin (proteolytic treatment) yields large and small
fragments, a single polypeptide of 68 kD (large fragment) which is known as the Klenow
fragment, lacks 5’ –3’ exonuclease activity and is used in in vitro replication.
• The small 35 kD (small) protein fragment contains the 5’-3’ exonuclease activity which
gives it the ability to start at a single-stranded break and progressively remove nucleotides
and replace them in a 5' to 3' direction
• The function of this polymerase during replication is to remove the RNA primers using a 5‘
3' exonuclease activity
• It then uses its 5‘ 3' polymerase activity to fill in the resulting gaps
• The primary functions to use the 5’-3’ exonuclease activity in removing the RNA primer
used during replication and fill in the gaps

Conti…
• If during polymerization an incorrect nucleotide is incorporated it is removed by the 3’ – 5’
exonuclease activity( i.e. it has proof reading activity)
• This give high fidelity, i.e. an error rate of less than 10-8 per base pair
• Although it is capable of template-directed DNA synthesis, it is now known not to be the
enzyme primarily responsible for new DNA synthesis

DNA Polymerase II
• The enzyme involved DNA repair
• 5‘- 3' polymerase activity and 3' to 5' exonuclease activity
• No 5' to 3' exonuclease activity.
• Uses DNA duplexes that have short gaps

DNA Polymerase III
• The primary enzyme involved in bacterial replication is DNA Pol III (DNA replication in vivo)
• It does the bulk of the DNA replication using its 5‘- 3' polymerase activity and 3' to 5' exonuclease
activity
• Pol III holoenzyme is an asymmetric dimer
• No 5' to 3' exonuclease activity.
• DNA polymerase III is a highly complex dimeric aggregate, consisting of 20 or more protein subunits
• Catalyses DNA syn @ 15000 bases/min at 300C (general assumption 1000 bases per second)

Theodor Hanekamp © 2003 8
DNA polymerase III holoenzyme



 
’

 
 
Core (
Linker
protein
Clamp
loader
“asymmetric dimer”
Source: “Model after A.Kornberg and T.Baker”,
adapted from Stryer
Polymerase
activity
3’- 5’
exonuclease
processivity
Core (

• Characteristics Pol I Pol II Pol III
• Gene pol A pol B pol C
• MW 103 kD 90 kD 130 kD
• molecules/cell 400 100 10
• 3’ - 5’ exonuclease act Yes Yes Yes
• 5’ – 3’ exonuclease act Yes No No
• Biol Function DNA repair SOS DNA replicative
repair growth

DNA polymerase structure
• Many DNA polymerases have a large cleft composed of three domains that
resemble a hand.
• DNA lies across the "palm" in a groove created by the "fingers" and "thumb.“
• The "palm" domain has important conserved sequence motifs that provide the
catalytic active site.
• The "fingers" are involved in positioning the template correctly at the active
site.
• The "thumb" binds the DNA as it exits the enzyme, and is important in
processivity.
• The most important conserved regions of each of these three domains
converge to form a continuous surface at the catalytic site.
• The exonuclease activity resides in an independent domain with its own
catalytic site.
• The N-terminal domain extends into the nuclease domain. DNA polymerases
fall into five families based on sequence homologies; the palm is well
conserved among them, but the thumb and fingers provide analogous
secondary structure elements from different sequences.

Primase (DnaG protein)
• This enzyme lays down the RNA primers that are necessary for DNA polymerase activity
• The requirement for a free 3’- OH group is fulfilled by the RNA primers that are synthesized at
the initiation site by this enzyme
• DnaG makes RNA primers (about 10 nucleotides long) that are used by DNA pol III
holoenzyme to start DNA synthesis
• Primase binds to dnaB protein at oriC and forms a primosome.
• The primase within the primosome complex provides RNA primers for synthesis of both
strands of duplex DNA.
• Primase lays down tracks of pppAC(N)7-10 (RNA).

Ligase
• Nicks occur in the developing DNA molecule because of the removal of RNA primer and synthesis
proceeds in a discontinuous manner on the lagging strand
• This enzyme seals nicks in DNA by linking up 3' –OH groups with adjacent 5' phosphate groups
• DNA Ligase will connect DNA to DNA but not DNA to RNA - so there is never a danger of RNA
primers being stitched into the nascent DNA
• The gaps in the discontinuous strand and also in the continuous strand are filled by the ligase

Facts check
1. What enzyme adds a DNA nucleotide to an existing nucleotide in DNA
elongation?
A. DNA polymerase III
B. DNA polymerase I
C. Primase
D. Helicase
E. DNA Ligase

What kind of primer does primase make?
A. DNA
B. RNA
C. Single-stranded binding protein
D. Ligated

What does helicase do?
A. Form phosphodiester bonds
B. Hold DNA strands apart
C. Replace RNA nucleotides with DNA nucleotides
D. Unwind & separate DNA strands

Why must DNA elongate in the 5’ to 3’ direction?
A. DNA ligase can only form covalent bonds at these Carbon locations
B. DNA Polymerase III can only add a nucleotide to the 3’ end of another
nucleotide
C. DNA Polymerase III can only add a nucleotide to the 5’ end of another
nucleotide
D. DNA Polymerase I can only add a nucleotide to the 5’ end of another
nucleotide
E. DNA Polymerase I can only add a nucleotide to the 3’ end of another
nucleotide

The Leading strand…
A. is formed in the 5’ to 3’ direction
B. is elongating into a replication fork
C. is elongating away from a replication fork
D. is composed of Okazaki fragments
E. Both A and B
F. Both C and D

DNA replication by Dr. YALAVARTHI NAGARAJU.pptx

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DNA replication by Dr. YALAVARTHI NAGARAJU.pptx