DNA Replication

Meselson and Stahl Experiment

Replicon and origin of Replicon

• DNA replication does not start at random sites but at particular sites,
called origin of DNA replication.

• A region in which replication starts from origin and proceeds

bidirectional or unidirectional to terminus site is called replicon, a unit
of DNA replication.

• A bacterial cell contains a unique origin and eukaryotic contains many

replication origins on single chromosome, hence many replicons.

• In E.coli single origin of the replication present in the chromosome is

referred to as oriC. The origin of replication is cis acting.

• oriC has 2 short repeat motifs, one of 9 and other 13 nucleotide long.

• The 9 nucleotide repeat is the binding site for a protein called DnaA.
The DnaA binding with the double helix, opens (melts) up within the
tandem array of 3 AT-rich from 13 nucleotide repeats.
• oriC contains 11 5'-GATC-3' repeats that are methylated on adenine
on both strands. Only fully methylated origin can start replication.

• Origin region contains AT-rich strech. This property facilitates

unwinding of DNA as less energy is required to break A-T bond.

• Origin of replication in yeast called autonomously replicating seq or

ARSs.

Replication Fork

• In E.coli, the replication fork proceeds at 1000 bp/sec/fork and 1

Okazaki fragment made in every 1-2 sec.

• In eukaryotes, the rate of fork movement is slow, ~ 100 bp/sec/fork.

DNA Helicase and Primase

• A DNA Helicase (DnaB) opens up the duplex at the replication fork to

provide a template. It moves on the lagging strand template in the
5'→3' direction unwinding the strand.

• This helicase requires ATP hydrolysis. The separated strands are

inhibited from binding to each other by single-stranded-binding-
protein (SSB protein).

• A primase synthesizes a short RNA primers to initiate DNA chain

elongation.

• The term Primosome used to denote complex between helicase and

primase.
Topoisomerase

• They relieve the positive supercoilig arises from

DNA unwinding mediated by helicase. All Topoisomerase
havenucleophilic
tyrosine
which use to
• The DNA gyrase is the 1st type II topoisomorase they
promote
has the ability to remove +ve supercoiling and Storand cleavage
introduce -ve supercoiling by using free energy
from ATP hydrolysis.

• DNA gyrase is the tetramer of 2 different subunits. GyrA subunit cut

and rejoin the DNA, while GyrB provides free energy by hydrolysis.
• All topoisomerase are of 2 classes- type I and type II.

• The type I cleave 1 DNA strand by passing other strand through the
break before resealing it. They do not require ATP as they get
energy stored from supercoiled DNA.

• The type II cleave 2 DNA strand by passing another double strand.

They do not require external energy source but utilizes ATP
hydrolysis to drive conformational changes in protein.

I
DNA Polymerase

• They catalyze the synthesis of DNA. They are of 2 types -

1. Template dependent
2. Template independent

• Template dependent are further classified into DNA dependent DNA

polymerase and RNA dependent DNA polymerase.

• DNA dependent DNA polymerase synthesizes a new DNA strand on

DNA template. RNA dependent DNA polymerase catalyzes the
synthesis of a new DNA strand on RNA template (Reverse
Transcriptase).

• Both prokaryotes and eukaryotes contain multiple DNA dependent DNA

polymerase. Some undertake replication and called DNA replicases.

• Prokaryotic DNA polymerases types -

DNA polymerase I -

• It is a monomeric protein composed of 928 amino acid residues.

• It posses 3 enzymatic activities -

1. 5'→ 3' polymerase activitity (independent of other two activities below)
2. 5'→ 3' exonuclease activity
3. 3'→ 5' exonuclease activity (proofreading)

• Proteases such as subtilisin or trypsin cleave Pol I into -

1. Larger C-terminal - Have polymerase and 3' → 5' exonuclease.
2. Smaller N-fragment - 5'→3' exonuclease activity.
• It has low processivity and low polymerisation rate (20 nucleotides/ sec)

DNA Polymerase III

• Primary enzyme involved in DNA replication and is a multiprotein

complex having 10 distinct polypeptides.

• It has a very high polymerization ( 1000 nucleotides/sec) and high

processivity ( > 500 Kb of DNA can continuously synthesized with DNA
polymerase dissociation from template).

• DNA Pol III has 4 components -

1. Catalytic core component - proofreading activity
2. Dimerization component - 2 catalytic core to form asymmetric dimer
3. Processitivity component
4. Clamp loader

• DNA Pol IV and V are Y- family DNA polymerase. They are only
involved in translesion and replicate damaged DNA by bypassing
damaged nucleotides.
• Eukaryotic DNA polymerases types -

DNA Polymerase @

• It is an unusual heterotetramer polymerase as it has both DNA

polymerase and primase activities. ( 5' → 3' DNA dependent DNA
polymerase and a 5'→ 3' DNA dependent RNA polymerase).

• With primase activity, it add NMPs (Nucleotide Monophosphates) to

synthesize short RNA primers ( 8-12 nucleotides) called initiator RNA
(iRNA) .

• With polymerase activity, it adds 20-30 dNMPs to 3' end of iRNA. The
DNA sometimes called initiator DNA (iDNA).

• It has no extrinsic 3'→5' exonuclease activity ( no proofreading) and

shows low processitivity.

• Later it is replaced by another DNA pol after iDNA. The process is

called as polymerase switching.

DNA polymerase delta and epsilon

g e

• Both these polymerase extend the DNA synthesis.

• DNA Pol delta in humans composed of 4-subunit. It has both 5→3'

endo and 3 '→ 5' exo activities.

• Pol delta requires associated 30 KDa protein called proliferating cell

nuclear antigen (PCNA), for high processitivity.
• The PCNA acts as a sliding clamp and requires a clamp loader
(Replication factor C).

• DNA Pol epsilon, a 4-subunit constituting nuclear enzyme having

Polymerase and Exonuclease activity. They carry out high processive
synthesis with the aid of sliding clamp and clamp loader and perhaps
the most accurate eukaryotic DNA.

• However the recent studies shows that Pol delta alone can replicate
leading and lagging strand without Pol epsilon.

E
Initiation

• The DnaA protein initiates replication in E.coli at oriC.

• DnaA binding to oriC-9 mers initiates strand separation of DNA duplex

at oric-13 mers. This process requires ATP by forming open complex.

• Further melting of strands caused by DnaB protein, a helicase.

Helicase (Hexamer) clamps around open complex on both strands. This
binding requires ATP and DnaC protein (Helicase loader).

• Helicase separates the strands using energy from ATP hydrolysis.

Further single stranded binding protein (SSB protein), helps separated
stands from joining again (reannealing).

• The primer makes short RNA sequences, whose synthesis catalyzed by

RNA polymerase primase. These RNA sequences are complementary to
DNA duplex. DNA polymerase attaches to primase for elongation.
Elongation

• DNA polymerase catalyzes the addition of deoxyribonucleotide units to

a DNA chain by nucleophilic attack from 3'-OH group of the primer.

• A catalytic metal ion Mg, reduces the 3'-OH group and makes
nucleophilic 3'-O. The Mg also neutralizes the -ve charge on incoming
dNTP and releasing pyrophosphate group (PPi).

• Subsequent PPi hydrolysis by pyrophosphatese (ubiquitous enzyme),

helps to drive polymerization forward.
• A leading strand synthesized continously from single primer in 5'→ 3'
in the same direction as of growing fork.

• After 1000-2000 nucleotides of the leading strand have replicated,

the first round of discontinuous strand synthesis on the lagging strand
can begin.

• Short DNA pieces called Okazaki fragments, repeatedly synthesized

on lagging strand by the process called semi-discontinuous replication.

• Okazaki fragment size are 1000-2000 nucleotide (bacteria) and

l0O-200 (eukaryotes).
 I

• The primase, associated with DnaB helicase, in the primosome, makes

RNA primer which extended by the DNA Pol III.

• In the lagging strand, the sliding clamp (Beta subunit) plays the role
of attachment and removal of DNA Pol III from lagging strand during
continuous formation of Okazaki fragments.

• By the completion of Okazaki fragments, the DNA Pol I enzyme

removes the primer and add subsequent DNA sequences. After that
the same DNA Pol I (Ligase) joins the gap between lagging strand.

• The DNA ligase catalyzes the formation of a phosphodiester bond

between the 3'-OH of one chain to 5'- phosphate group of other
chain.

• The energy source used as ATP for ligase in archae and eubacteria.
• The ATP or NAD+ as the AMP donor during the reaction by DNA ligase
and PPi or NMN is
released.

• In eukaryotes, the RNA primer removal is done by RNase H because no

eukay polymerase shows any 5'→ 3' exonuclease activity.

• The Rnase H can degrade the RNA part of base paired RNA-DNA
hybrid, but cannot cleave the phosphodiester bond between the
last Ribonucleotide and first Deoxyribonucleotide. The last bond thus
 cleaved by flap endonuclease (FEN1).

Termination

• Bacterial genomes are replicated bidirectionally from a single point

and meets at the diametrically opposite end on genome map.

• Termination occurs on 23 bp sequences called Ter sequence (terminus).

7 of these sequences are identified and each acts as recognition site
for DNA-binding protein called Tus ( terminus utilization substance).

• Two replication forks moving in opposite direction when reaches the

Tus protein, the Tus blocks the replication of One fork by blocking its
Dna B (Helicase) passage through it while allowing the other fork's.

• Thus both the replication fork become trapped within a relatively

short region opposite to origin (OriC).

D
• Then both the catenated (interlocked) DNA circles are decatended by
topoisomerase IV (type II topo).

Telomere
E
Replication

• All eukaryotic
chromosomes end in
telomeres.

• The replication process of

telomere repeats with the
help of special enzyme,
telomerase, is independent of normal DNA replication.

• The telomerase resembles the reverse transcriptases, which

synthesize DNA using RNA template.

• Telomerase first synthesize a copy of repeat using its own RNA

 template component in the G-rich strand of telomeric DNA in 5'→ 3'.

• After the extension of leading strand, the lagging strand replication is

done using leading strand extension as
template and RNA primer and
polymerase in 5'→ 3'.

Replication of
mitochondrial DNA
It

• Small and mostly circular mitochondrial

and chloroplast DNA use slightly
different process duplication.

• In this case, a primer 1st binds to one

of the strand and initiates the
replication and removes primer after
initiation.

• As the new DNA being synthesized,

that creates a displacement or D-loop. Some mitochondrial DNA have
several loops showing multiple origins.

• DNA polymerase is used.The replisome machinery formed by DNA

poly, TWINKLE (Helicase), and SSB protein.
Rolling
Circle DNA
Replica Replication
tion