Prokaryotic and Eukaryotic

DNA Replication UST Faculty of Pharmacy

Department of Biochemistry
DNA Replication

•Biosynthesis of Nucleic Acids

•How do cells duplicate their contents?
•DNA duplicates by a process called DNA
replication.
Flow of Genetic Information in the Cell
• Mechanisms by which information is transferred in
the cell is based on “Central Dogma”
Three Models of DNA Replication
Figure 1. Experiment by
Meselson and Stahl
Parental DNA (15N-labeled or
heavy DNA) is replicated
semiconservatively so that in first
generation, DNA molecules
contain one parental (blue) strand
and one newly synthesized (red)
strand.
In succeeding generations, the
proportion of 14N-labeled or light
strands increases, but hybrid
molecules containing one heavy
and one light strand persist.
 DNA replication
• Takes place by separation of the strands of the double helix,
and synthesis of two daughter strands complementary to
the two parental templates.
• Unwinding and separation of the two DNA strands
• Protection of unwound portions from attack by nucleases
that attack single-stranded DNA
• Synthesis of the DNA template from one 5’ -> 3’ strand and
one 3’ -> 5’ strand
• Efficient protection from errors in replication.
 Overview of DNA Replication
DNA-directed DNA polymerases – enzymes that catalyze the biosynthesis of exact copy of
DNA by using a single-strand DNA (ssDNA) as template. DNA chains are extended only in the
5’ to 3’ direction which is driven by the subsequent hydrolysis of pyrophosphate.
General Features of DNA Replication
• Each strand can serve as template.
• Semi-conservative
• Requires primers
• Semi-discontinuous
• Usually bidirectional
• Ordered, sequential and highly accurate
DNA polymerase
•DNA-directed DNA polymerase is responsible for
synthesizing new DNA strands from a DNA
template.
•DNA polymerase requires a primer which
provides the 3’–OH terminus on which to add
new nucleotides.
•Polymerization occurs in the 5’-to-3’ direction.
DNA Replication Occurs at Replication Forks

θ structures – circular structures

in the chromosome referred to as
replication eye or bubbles that
formed when two strands of DNA
unwind to initiate replication.
Replication fork – branch point in
replication eye where the DNA
synthesis occurs. N of replication
fork determines whether DNA
synthesis is unidirectional or
bidirectional.
Features
• Both daughter strands are synthesized simultaneously.
Both strands replicated in the 5’- 3’ direction.
• The leading strand is synthesized continuously in the
5’ -> 3’ direction toward the replication fork.
• The lagging strand is synthesized discontinuously
(Okazaki fragments) also in the 5’ -> 3’ direction, but
away from the replication fork.
DNA Replication is Semidiscontinuous
Okazaki fragments (Reiji Ozaka) – newly
synthesized DNA sequences spanning 100 to 200
nt long which is covalently joined into a larger
DNA molecule.
Semidiscontinuous – two parent strands are
synthesized in different ways: leading strand is
continuously synthesized at 5’ to 3’ direction
while lagging strand is also directed in the same
direction but made discontinuously.
DNA ligase – enzyme that connects Okazaki
fragments to lagging strand DNA during synthesis
RNA primers – produced by the enzyme primase
which are RNA segments of 1 to 60 nt long
located in the 5’ends of growing DNA chain.
DNA Replication is bidirectional
• DNA double helix unwinds at a specific point called an
origin of replication.
• Polynucleotide chains are synthesized in both
directions from the origin of replication; replication
proceeds bidirectionally.
• At each origin of replication, there are two replication
forks, points at which new polynucleotide chains are
formed.
 Prokaryotic DNA Replication: E. coli

Prokaryotes or Bacterial cells divide by Binary fission. The genetic material is circular compared
to linear structure of DNA in eukaryotes. But, the Replication process is the same which is
initiated from a single point of origin of replication and proceeds in opposite direction.
• Recognition of origin of replication by specific proteins DnaA binding
results to “melting” of the double helix at the origin of replication.
• Formation of replication fork
• Helicase, a helix-destabilizing protein, promotes unwinding by binding
at the replication fork
• Single-stranded binding (SSBs) proteins stabilize single-stranded
regions by binding tightly to them
• Primase (RNA polymerase that catalyzes the copying of a short
stretch of the DNA template strand to produce RNA primer
sequence), helicase and SSB proteins form a“primosome”, which
processively moves along the lagging-strand template.
• The two tethered polymerases can replicate both strands by
looping the DNA of the lagging-strand template back on
itself, causing this template to havethe same orientation as
the leading-strand template.

• Once the polymerase assembling the lagging strand reaches

the 5’ end of the Okazaki fragment synthesized during the
previous round, the lagging-strand template is released and
the polymerase begins work at the 3’ end of the next RNA
primer toward the fork.
 DNA Polymerases of E. coli

Speed of the reaction – turnover number

Processivity – it refers to the number of nucleotides joined before the enzyme
dissociate from the template.
DNA Polymerase III: Major DNA Replication Enzyme

The catalytic core of the enzyme (α, θ, and ε) and seven other subunits form the labile
multisubunit Pol III holoenzyme. The enzyme can synthesize DNA strand
complementary to a ssDNA template and can edit the polymerization reaction (fidelity).
• Excision of RNA primers by DNA polymerase I
• 5’-3’ exonuclease activity function removes
approximately 10 nucleotides from the 5’ end of a
single strand nick. This activity plays a key role in
removing the RNA primer.
• Filling up of the gaps by DNA polymerase I
• Nick ligation by DNA ligase
 Requirements for Successful DNA Replication
§ Primer in DNA replication – short stretch of RNA hydrogen-bonded to the template
DNA to which the growing DNA strand is bonded at the start of synthesis.
§ Four deoxyribonucleoside triphosphates – dTTP, dATP, dGTP and dCTP
§ Mg+2 – metal cofactor
§ DNA template
§ Exonuclease activities – proofreading-and-repair activities of DNA replication
proofreading – the process of removing incorrect nucleotides when DNA
replication is in progress
repair – the enzymatic removal of incorrect nucleotides from DNA and their
replacement by correct ones
§ Replisome – multiprotein complex consists of DNA polymerase, RNA primer, primase
and helicase at the replication fork
 Supercoiling and Replication

Figure 2. DNA Gyrase introduces supertwisting in circular DNA.

DNA Gyrase – an enzyme that introduces supercoiling into closed circular DNA
Mechanism of DNA Replication starting from Replication fork

Figure 3. General features of a replication fork.

Helicase (DnaB) – an enzyme that catalyze the unwinding of the double helix of DNA in the process or replication.
Single-strand binding protein (SSB) – protein that protects exposed single-strand sections of DNA from nucleases.
Primase – the enzyme that makes a short section of RNA to act as a primer for DNA synthesis.
Summary of Prokaryotic DNA Replication Mechanism
Termination of Replication
• Terminator sequences: recognition site for a sequence-specific DNA-
binding protein called Tus protein.
• Orientation of bound Tus relies on the orientation of the termination
sequences.
• Tus protein allows a replication fork to pass if the fork is moving in
one direction, but blocks progress if the fork is moving in the opposite
direction around the genome.
Summary of Prokaryotic DNA Replication Mechanism
1. DNA synthesis is bidirectional. Two replication forks advance in opposite directions from an origin of replication.
2. The direction of DNA synthesis is from 5' end to the 3' end of the newly formed strand. One strand (the leading
strand) is formed continuously, while the other strand (the lagging strand) is formed discontinuously. On the
lagging strand, small fragments of DNA (Okazaki fragments) are subsequently linked.
3. Five DNA polymerases have been found in E. coli. Polymerase III is primarily responsible for the synthesis of
new strands. The first polymerase enzyme discovered, polymerase I, is involved in synthesis, proofreading, and
repair. Polymerase II, IV, and V function as repair enzymes under unique conditions.
4. DNA gyrase introduces a swivel point in advance of the movement of the replication fork. A helix-destabilizing
protein, a helicase, binds at the replication fork and promotes unwinding. The exposed single-stranded regions of
the template DNA are stabilized by a DNA-binding protein.
5. Primase catalyzes the synthesis of an RNA primer.
6. The synthesis of new strands is catalyzed by Pol III. The primer is removed by Pol I, which also replaces the
primer with deoxynucleotides. DNA ligase seals the remaining nicks.
Proofreading and Repair Functions of DNA Polymerase

Figure 4. DNA polymerase 3’ to 5’ proofreading mechanism.

Mutations – errors in replication machinery. It can be induced spontaneously during the process of
DNA synthesis or can be induced by exposure to mutagens.
DNA Pol I has three active sites (Hans Klenow) which can be cleaved to 2 major fragments: (1) Klenow
fragment contains the polymerase and proofreading activity and (2) contains the 5’ to 3’ repair
activity
Proofreading and Repair Functions of DNA Polymerase
Nick translation – the cut-and-
patch process catalyzed by
DNA Pol I where the cut
involves removal of RNA
primers or the removal of
mismatched or damaged
nucleotides by 5’ to 3’
exonuclease activity and
rectify the newly synthesized
strand by incorporating the
correct nucleotides to fill in
the gap by its polymerase
activity.
Mismatch repair – a type of
DNA repair that begins when
Figure 5. DNA repair enzymes find two bases
polymerase 5’ to 3’ that are incorrectly paired.
repair activity.
 Eukaryotic DNA Replication
DNA replication occurs only ONCE per cycle of cell division.
Four stages of Cell Division
1. M, Mitosis – division of the nucleus or when replicated chromosomes
are separated into two nuclei. This stage is preceded by interphase of S
phase followed by cytokinesis, the division of cells into two equal
components .
2. G1, Gap 1 – longest part of the cell cycle where rapid growth and
metabolic activity of the cells take place
3. S, Synthesis phase – time of DNA synthesis
4. G2, Gap 2 – relatively short period of growth in which the cell prepares
for division.
Replicators – the multiple origins of replication in eukaryotic DNA
synthesis
Replicons – parts of chromosomes in which DNA synthesis is taking place
Origin recognition complex (ORC) – a protein complex bound to DNA
Figure 6. The Eukaryotic Cell Cycle. throughout the cell cycle that serves as an attachment site for several
proteins that helps control replication.
 Figure 7. Model for the initiation of the
DNA replication cycle in eukaryotes.

Replication activator protein (RAP) – the protein whose

binding prepares for the start of DNA replication in
eukaryotes
Replication licensing factors (RLFs) – proteins required
for DNA replication in eukaryotes
Pre-replication complex (pre-RC) – the complex of DNA,
recognition protein (ORC), RAP, and RLFs that makes DNA
competent for replication in eukaryotes
Cyclins – proteins that play key roles in control of the cell
cycle by regulating activity of kinases
Cyclin-dependent protein kinases – protein kinases that
interact with cyclins and control replication
 Eukaryotic DNA Polymerases

Figure 8. Structure of the PCNA homotrimer

Polymerase δ is the principal DNA polymerase in eukaryotes.

It interacts with PCNA (proliferating cell nuclear antigen),
which acts as sliding clamp that surround DNA. Separate
exonucleolytic enzymes exist in animal cells for repair.
Eukaryotic vs Prokaryotic DNA Polymerase
Key Features of Eukaryotic DNA
replication:
§ The primase activity is associated with
DNA Pol α
§ Formation of smaller Okazaki fragments
is initiated by DNA Pol α
§ After RNA primer is made, Pol α adds
few nucleotides, the polymerase
dissociates and replaced by Pol δ and
PCNA protein
§ Replication factor C (RFC) is involved in
binding Pol δ-PCNA complex
§ Don’t have 5’ to 3’ exonuclease activity
but has separate enzymes, FEN-1 and
RNase H1 that degrade RNA
§ Continued movement of Pol δ fills in the
gaps made by primer removal
§ Histones (not present in prokaryotes)
biosynthesis occur at the same time as
DNA replication
Mechanism of Eukaryotic DNA Replication

Figure 9. The basics of the eukaryotic replication fork.