DNA REPLICATION

Assoc. Prof. Dr. Ilke ONEN

 DNA replication is the process by which DNA makes a copy of
itself during cell division
Semiconservative DNA Replication

Watson and Crick DNA model implies

a mechanism for replication:
a. Unwind the DNA molecule.
b. Separate the two strands.
c. Make a complementary copy for each
strand.
DNA POLYMERASES, THE DNA REPLICATING ENZYMES
First isolation of an enzyme involved in DNA replication was in
1955. Arthur Kornberg won the 1959 Nobel Prize in
Physiology or Medicine for this work.
Accomplished in vitro synthesis of E. coli DNA. His reaction
mixture included:
a. DNA fragments (template).
b. Radioactively labeled dNTPs (dATP, dGTP, dTTP and
dCTP).
c. E. coli lysate.

Enzyme originally called the Kornberg enzyme, now known as

DNA Polymerase I.

Once isolated, could characterize its activity, showing that

the above components are required, along with Mg2+ ions
for maximum activity
 ROLES OF DNA POLYMERASES
DNA chain elongation is catalyzed by DNA polymerase. DNA polymerases synthesize only from 5’ to 3’.
 PROKARYOTC DNA POLYMERASES

Polymerase 5'→3' Polymerase 3'→5' Exonuclease 5'→3' Exonuclease

activity activity activity

DNA Polymerase I + + +

DNA Polymerase II + + -
DNA Polymerase III + + -

DNA Polymerase IV + - -
DNA Polymerase V + - -
Properties and structure of DNA Polymerases
Properties and structure of DNA Polymerases

DNA polymerases are capable of adding as many as

1000 nucleotides/sec to a primer strand.

The speed of DNA synthesis is largely due to the

processive nature of DNA polymerase.

In the case of DNA polymerases, the degree of

processivity is defined as the average number of
nucleotides added each time the enzyme binds a
primer:template junction.
Correctly paired bases are required for DNA-polymerase-catalyzed nucleotide addition.
3’ 5’ EXONUCLEASE ACTIVITY
(PROOFREADING)

 3’ 5’ Exonuclease activity
(Proofreading): Removal of
incorrect base
DNA POLYMERASE III HOLO ENZYME

•The DNA Pol III holoenzyme includes two copies of the “core” DNA Pol III enzyme
and one copy of the five subunit sliding clamp loader.

•The sliding clamp loader includes two copies of tau (t) protein, each of which
binds one DNA Pol III core enzyme.
Sliding clamp
Sliding DNA clamps encircle the newly replicated DNA produced by an associated DNA
polymerase. The sliding clamp interacts with the part of the DNA polymerase that is closest to the
newly synthesized DNA as it emerges from the DNA polymerase.

Sliding DNA clamps increase the processivity of associated DNA polymerases.

After DNA polymerase has completed synthesis of the template, the absence of a
primer:template junction causes a change in the DNA polymerase that releases it from the sliding
clamp.
Eukaryotic Replication Enzymes
Initiation of Replication
 INITIATION OF REPLICATION

The specific sites at which DNA unwinding and initiation of

replication occur are called origins of replication.
When DNA denatures at the origin of replication, 2 Y-shaped
structures (replication forks) are formed, together making up
what's called a replication bubble.

DNA replication is usually bi-directional, but will consider

events at just one replication fork.
 REPLICON IN PROKARYOTES

 In 1963, three scientists proposed a model to explain the events controlling the
initiation of replication in bacteria.
 They defined all of the DNA replicated from a particular origin of replication as a
replicon.
 For example, because the single chromosome found in E. coli cells has only one
origin of replication, the entire chromosome is a single replicon.
INITIATION DNA SYNTHESIS IN E. COLI
E. coli has one origin, oriC, which has:
a. A minimal sequence of about 245 bp
required for initiation.
b. Three copies of a 13-bp AT-rich sequence.
c. Four copies of a 9-bp sequence.

Initiator proteins recognizes this sequences and

opens the double helix. E. coli’s initiator protein is
DnaA.
 DNA HELICASE

 Once the DNA has been opened at the

origin, the enzyme DNA helicase catalyzes
the breaking of the hydrogen bonds
between the base pairs using ATP as an
energy source.
SINGLE-STRAND DNA-BINDING PROTEINS (SSBS)
Single-strand DNA-binding proteins
(SSBs) bind the ssDNA molecules formed
by helicase, preventing them from
rejoining each other (reannealing).
Binding of one SSB promotes the binding
of another SSB to the immediately
adjacent ssDNA.
Primase

All DNA polymerases require a primer with a free 3’-OH.

They cannot initiate a new DNA strand de novo. To accomplish this, the
cell takes advantage of the ability of RNA polymerases to do what DNA
polymerases cannot: start new RNA chains de novo.

Primase, an RNA polymerase that builds strands of RNA. Primase can

start a strand on its own and it makes short pieces of RNA (5–10nt),
called primers that are complementary to the DNA.

These primers are subsequently extended by DNA polymerase.

Helicase + Primase = Primosome complex

MODEL FOR THE FORMATION OF A REPLICATION BUBBLE AT A REPLICATION ORIGIN IN
E. COLI AND THE INITIATION OF THE NEW DNA STRAND
Elongation of Replication
Leading and Lagging Strands

New strand made 5’ → 3’ in same direction as movement of the replication fork is leading strand,
while new strand made in opposite direction is lagging strand. Leading strand needs only one
primer, while lagging needs a series of primers.
Leading strand is synthesized continuously, while lagging strand is synthesized discontinuously, in
the form of Okazaki fragments. DNA replication is therefore semidiscontinuous.
5’-3’ template DNA probably bends to allow synthesis of both leading and lagging
strands at the same time.
Model for the Events Occurring Around a Single Replication
Fork of the E. Coli Chromosome
 Model for the Events Occurring Around a Single Replication
Fork of the E. Coli Chromosome

Okazaki data show that these fragments are gradually joined together to make a full-length
dsDNA chromosome.
DNA polymerase I uses the 3’-OH of the adjacent DNA fragment as a primer, and removes the
RNA primer (due to its 5'-3' exonuclease activity) while resynthesizing the primer region in the
form of DNA.
DNA ligase catalyzes the last phosphodiester bond.
 DNA ligase

DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing
the formation of a phosphodiester bond between a 3′-OH group and a 5′-monophosphate.
Formation of Supercoils

Unwinding of the helix during DNA

replication (by the action of helicase) results
in supercoiling of the DNA ahead of the
replication fork.

This supercoiling increases with the

progression of the replication fork.

If the supercoiling is not relieved, it will

physically prevent the movement of helicase.
DNA topoisomerases

 DNA topoisomerases are a class of enzymes involved in the regulation of DNA

supercoiling. Type I topoisomerases change the degree of supercoiling of DNA by
causing single-strand breaks and re-ligation, whereas type II topoisomerases (such
as bacterial gyrase) cause double-strand breaks.
 DNA helix must be unwound to allow proper function of large enzymatic
machinery, and topoisomerases have indeed been shown to maintain replication.
Termination of Replication
 Termination of Replication in Prokaryotes

Two replication forks of E. Coli meet at terminus

regions (ter)

There are as many as 10 terminator sequences

on the E. Coli chromosomes.

Terminal utilization substance(Tus) proteins bind

to terminator sequences and inhibit helicases.

Replication fork can pass through the Tus-DNA

complex in one direction but not the other.

Topoisomerase IV is responsible for separating

newly replicated DNA molecules
 On average, DNA polymerase
inserts one incorrect nucleotide
for every 105 nucleotides added.
 Proofreading exonucleases
decrease the appearance of
incorrect base pairs to 1 in every
107 nucleotides added.
 The actual rate of mutation
observed in a typical cell
(approximately one mistake in
every 1010 nucleotides added.
(due to mismatch repair system)
DNA Replication in Eukaryotes
DNA replication is very similar in both prokaryotes and eukaryotes, except that eukaryotes have
more than one chromosome

Eukaryotic origins are generally not well characterized; those of the yeast Saccharomyces
cerevisiae are among the best understood.

Chromosomal DNA fragments (about 100bp) that are able to replicate autonomously when
introduced into yeast as extracellular, circular DNA are known as ARSs (autonomously replicating
sequences). ARSs are yeast replicators.

Eukaryotic replication forks move much more slowly. In eukaryotes, replicon size is smaller than it is
in prokaryotes.

Replicating DNA of Drosophila melanogaster

 DNA Replication in Eukaryotes

Initiation of replication has two separate steps, controlled

by cyclin-dependent kinases (Cdks) that are present
throughout the cell cycle, except during G1.

In the absence of Cdks during G1, replicator selection

occurs. ORC and other proteins assemble on each
replicator to from pre-replicative complexes (pre-RC).

When cell enters S phase, Cdks are present, and activate

pre-RCs to initiate replication.

Cdk activity inhibits another round of pre-RC formation

until the cell again enters G1, when Cdks are absent
 Prokaryotic Replication vs Eukaryotic Replication

Prokaryotes Eukaryotes
Helicase Mini chromosome maintenance (MCM 2-7)

SSB Replication protein A (RPA)

Sliding clamp (β-clamp) Proliferating cell nuclear antigen (PCNA)
Okazaki fragments are 1000–2000 bases in Okazaki fragments are 100–400 bases in length
length
Primers are 10 bases in length Primers are 100 bases in length

Two molecules of DNA polymerase III carry out DNA polymerase delta (δ) is responsible for
coordinated synthesis of both the leading and synthesis lagging strand
lagging strands of DNA DNA polymerase epsilon (ε) is responsible for
synthesis leading strand
The Problem of Replicating Completely a Linear
Chromosome in Eukaryotes

When the ends of chromosomes are replicated and the

primers are removed from the 5’ ends, there is no adjacent
DNA strand to serve as a primer, and so a single-stranded
region is left at the 5’ end of the new strands.

If the gap is not addressed, chromosomes would become

shorter with each round of replication.
 Telomeres

The terminal regions of eukaryotic chromosomes are

organized as a specialized structure to protect and
maintain the stability of the chromosomes.

Telomeres in most eukaryotes consist of short, tandemly

repeated DNA sequences, such as (TTTGGG)n in
humans .

Telomere length may vary, but organisms and cell

types have characteristic telomere lengths.

Telomere length is genetically controlled.

 Telomerase

Blackburn and Greider have shown that chromosome

lengths are maintained by telomerase, which adds
telomere repeats without using the cell’s regular
replication machinery.

Telomerase is active in gametes and most cancer

cells, but is normally absent from, or at very low levels
in, most somatic cells.
Telomerase

Telomerase carries its own RNA molecule (TR)

which is used as a template when it elongates
telomeres

Telomerase reverse transcriptase (TERT) is a

reverse transcriptase, which is a class of enzyme
that specifically elongates the 3’-OH of telomeric
ssDNA sequences using its own RNA as a template.

As a result of this unusual mechanism, the newly

synthesized DNA is single-stranded.
SYNTHESIS OF TELOMERIC DNA BY TELOMERASE

G strand
C strand (parental strand)
(new strand) telomerase
RNA (TR)
telomerase

TERT can add a six-nucleotide

repeating sequence

Telomerase only directly extends the 3’

end of the telomere (G strand), by
providing an additional template for
lagging-strand DNA synthesis, both ends
of the chromosome are extended.
 Telomerase
relocates

still has 3’
overhang

DNA
synthesis RNA primer polymerase
direction
 T-loop formation

Once the ssDNA end is positioned properly,

it can invade the dsDNA repeats and form a
helix with the complementary strand,
displacing the other strand of the dsDNA.

This is called a displacement loop.

Proteins bound to telomeric DNA act to

regulate the activity of telomerase and
protect the ends of chromosomes from
degradation and recombination.