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DNA replication
Learning objectives
Understand the basic rules governing
DNA replication
Introduce proteins that are typically
involved in generalised replication
Reference: Any of the recommended texts
http://www.dnai.org/lesson/go/2166/1973
Optional
Nature (2003) vol 421,pp431-435
http://www.bath.ac.uk/bio-sci/cbt/
`It has not escaped our notice that the
specific pairing we have postulated
immediately suggests a possible
copying mechanism for the genetic
material’
Watson & Crick
Nature (1953)
Original drawing by Francis Crick
Four requirements for DNA to
be genetic material
Must carry information

Cracking the genetic code
Must replicate

DNA replication
Must allow for information to change

Mutation
Must govern the expression of the
phenotype

Gene function
DNA stores information in the sequence of
its bases
Much of DNA’s sequence-specific information is
accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as
the DNA helix unwinds. Proteins can either bind to a
DNA sequence, or initiate the copying of it.
Some genetic information is accessible even in
intact, double-stranded DNA molecules
Some proteins recognize the base sequence of DNA
without unwinding it.
One example is a restriction enzyme.
DNA Replication
Process of duplication of the entire genome prior to cell
division
Biological significance

extreme accuracy of DNA replication is necessary in
order to preserve the integrity of the genome in
successive generations

In eukaryotes , replication only occurs during the S
phase of the cell cycle. The slower replication rate in
eukaryotes results in a higher fidelity or accuracy of
replication in eukaryotes
Basic rules of replication
A.
B.
C.
D.
E.
F.
Semi-conservative
Starts at the ‘origin’
Can be uni or bidirectional
Semi-discontinuous
Involves DNA templating
RNA primers required
A) DNA replication – three possible
models



Semiconservative replication –
Watson and Crick model
Conservative replication: The parental
double helix remains intact; both
strands of the daughter double helix
are newly synthesized
Dispersive replication: At completion,
both strands of both double helices
contain both original and newly
synthesized material.
Meselson-Stahl experiments confirm
semiconservative replication
Semi-conservative replication An
ingenious experiment by Meselson and
Stahl showed that one strand of each
DNA strand is passed on unchanged to
each of the daughter cells. This
'conserved' strand acts as a template for
the synthesis of a new, complementary
strand by the enzyme DNA polymerase
Meselson-Stahl experiments confirm
semiconservative replication
Double helix
unwinds
Each strand acts
as template
Complementary
base pairing
Two daughter
helices produced
after replication
Starts at origin
Initiator proteins identify specific base
sequences on DNA
Prokaryotes – single ori site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes
Eukaryotes
Uni or bidirectional
Bidirectional replication





Replication forks move in opposite
directions
In linear chromosomes, telomeres ensure
the maintenance and accurate replication
of chromosome ends
In circular chromosomes, such as E. coli,
there is only one origin of replication.
In circular chromosomes, unwinding and
replication causes supercoiling, which may
impede replication
Topoisomerase – enzyme that relaxes
supercoils by nicking strands
The bidirectional replication of a
circular chromosome
Semi-discontinuous replication
Anti parallel strands replicated simultaneously
 Leading strand synthesis continuously in 5’– 3’
 Lagging strand synthesis in fragments in 5’-3’
Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction
DNA templating
Nucleotide recognition
Enzyme catalysed
polymerisation
Complementary base
pair copied
Substrate used is dNTP
RNA primers required
Core proteins at the replication fork
Topoisomerases
Helicases
Single strand
binding proteins
Primase
DNA polymerase
Tethering protein
DNA ligase
- Prevents torsion by DNA breaks
- separates 2 strands
- prevent reannealing
of single strands
- RNA primer synthesis
- synthesis of new strand
- stabilises polymerase
- seals nick via phosphodiester
linkage
The mechanism of DNA replication
Arthur Kornberg, a Nobel prize winner and
other biochemists deduced steps of
replication

Initiation



Elongation


Proteins bind to DNA and open up double helix
Prepare DNA for complementary base pairing
Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
Termination

Proteins release the replication complex
Core proteins at the replication fork
Nature (2003) vol 421,pp431-435
Figure in ‘Big’ Alberts too
Topoisomerase I
Cause temporary
single strand breaks
in DNA
Allows free rotation
of helix
Monomeric enzymes
(~100kD)
Reversible
catenation (knotting)
Acts by breaking
phosphodiester
linkage via tyrosine
active site
Topoisomerase II (DNA gyrase)
Cause temporary double strand breaks in DNA
Topoisomerase II (DNA gyrase)
~ 375 kD
2 pairs of subunits:
A&B
ATP hydrolysis
mediated double
strand break
Catenates double
stranded circles
Inhibited by
antibiotics