25| DNA Metabolism

2013 W. H. Freeman and Company

DNA replication
Duplication of a single DNA molecule into two identical copies.
A unique property of DNA that makes it a suitable genetic
material.
Replication of DNA occurs with high fidelity.
This is possible because two strands of DNA are
complementary to each other.
Due to this complementarity, two strands of DNA serve as the
template for the synthesis of new strands with a predictable
and complementary sequence.
The fundamental property of DNA replication process and the
mechanism used by the enzymes that catalyze it is essentially
identical in all species.

DNA replication is semiconservative

Hypothesized by Watson and Crick.
Proved by Meselson and Stahl.

Meselson-Stahl Experiment

Meselson-Stahl Experiment

Meselson-Stahl Experiment

NA is replicated during S phase of the cell cyc

Replication begins at an origin

Replication always initiate at a unique point called origin.
Origin is an AT-rich region.
Bacterial chromosome has single origin of replication.

Eukaryotic chromosomes have multiple

origins of replication that are spread
out across the chromosome length
Origin of replication in eukaryotes are called ARS

Origins of replication are strictly controlled so

that they fire only once per cell cycle.

Each origin of replication initiates the

formation of bidirectional replication
forks

DNA synthesis always proceed in 5 a 3

direction

Both Template strands are copied at a replication fork

DNA
Polymerization

Replication fork has leading and lagging strands

The polarity of DNA synthesis results in continuous synthesis of
DNA on
one strand and discontinuous synthesis on the other strand.
The strand that is synthesized continuously has its growing 3
end in the
replication fork and is called leading strand.
The strand that is synthesized discontinuously has its growing 3end away
from the replication fork and is called lagging strand.
Lagging strand is synthesized in the form of small fragments
called
Okazaki fragments (1000-2000 nt long in bacteria and 150 nt long in higher
eukaryotes).

DNA synthesis is semidiscontinuous

DNA is synthesized by DNA polymerases

DNA polymerases of E. Coli

DNA
Polymerization

DNA Synthesis

DNA Synthesis

Hydrolysis of
pyrophosphate
drives DNA
2Pi synthesis
H2O

Fidelity of Replication
Error rate is less than 1 in 10 8
(1) Complementary base pairing
(2) Proofreading activity of DNA
polymerase

Error correction by 3a5

exonuclease activity proofreading

DNA polymerases of E. Coli

DNA replicase system : Replisome

DNA polymerase III and about 20 other different proteins required for
replication of DNA in E. Coli.

Stages of DNA Replication

Initiation
Elongation
Termination

E. coli replication origin

DNA unwinding element

(DUE)

R sites

Initiation

+ve supercoil
8

Elongation

Elongation

Elongation

Elongation

DNA polymerase catalytic

subunit is not very
processive (i.e. it falls
off the DNA easily).
A sliding clamp ()
is required to keep
catalytic subunit on and
allow duplication of long
stretches of DNA

A clamp loader: (, , )
complex is required
to get the clamp onto
the DNA

Lagging strand synthesis

Topoisomerase

PCNA

MCM
proteins

RPA
RFC

DNA polymerase III dimer

Subunits of DNA polymerase III of E. Coli

Coordination of Synthesis

Final Stages of Elongation

DNA Ligase Reaction

Termination at Circular
Chromosomes

Separation of
Strands