7.

Replication
Replication origins and forks, primers, telomeres.
8. Repair and Recombination
Mutation and repair, homologous recombination.

Alberts, et al., Essential Cell Biology

Chapter 6.
 DNA Replication
Cells do not live forever, and in light of this,
they must pass their genetic information on to
new cells, and be able to replicate the DNA to
be passed on to offspring.

DNA is replicated by uncoiling of the helix,

strand separation by breaking of the hydrogen
bonds between the complementary strands,
and synthesis of two new strands by
complementary base pairing.

Since each of the new daughter molecules

contains one new and one old strand DNA
replication is said to be semi-conservative.
3
4
 DNA Replication
New complementary strands are produced by 5' 3'
the hydrogen bonding of free DNA
nucleotides with those on each parent strand.

daughter
strand
As the new nucleotides line up opposite each O
parent strand by hydrogen bonding, enzymes

Template strand
O P O- Base

called DNA polymerases join the nucleotides O

by way of phosphodiester bonds. H
O
H

H H
OH H
The nucleotides lining up by complementary O -
O -

base pairing are deoxynucleoside

Base
-
O P O P O P
O-
triphosphates. O O O-
O
H H

As the phosphodiester bond forms between

H H
OH H

the 5' phosphate group of the new nucleotide O- O-

O
Base

and the 3' OH of the last nucleotide in the

-
O P O P O P 5'
O-
5'
DNA strand, two of the phosphates are O O O-
O

removed providing energy for bonding. H H

H H
OH H
dNTP
Deoxynucleotide triphosphate
DNA replication begins at an origin where the double helix separates
into single strands.

This creates 2 replication forks that move away from each other as the
DNA is copied (replication is bidirectional).

An enzyme called DNA helicase unwinds the DNA at the replication

fork using energy from ATP hydrolysis.

Synthesis of the new DNA chain takes place in the 5’ to 3’ direction

only.
7
11
 DNA replication is semi-discontinous
3’
3’ 5’ Leading strand
5’
Okazaki fragments
3’
5’ 3’ Lagging strand
5’

The antiparallel structure of DNA poses a problem for DNA replication. As the replication
fork advances daughter DNA (newly synthesised) strands must be synthesised on both of
the exposed parental stands. The replication fork is moving in the 5’ to 3’ direction on one
strand and in the 3’ to 5’ direction on the other. However, DNA polymerases only add a
nucleotide to the free 3'-OH group of a base paired polynucleotide, so that DNA chains are
extended only in the 5' to 3' direction. The problem is solved by synthesising the 3’ to 5’
strand in a series of fragments synthesised in the 5’ to 3’ direction.

DNA polymerases need a partly double stranded structure to work on, so a primase
synthesises a short stretch of RNA - about 10 nucleotides called the primer - so the DNA
polymerase can get started.
13
 Proteins involved in the replication fork
New strand
DNA polymerase
single-stranded DNA
leading binding proteins
template
helicase
lagging template

DNA Parent duplex

primase
DNA Okazaki
RNA primer
polymerase fragment Direction of movement
of fork along parent DNA
 Replication fork

DNA helicase
hydrolyses ATP and
uses the energy to
unwind the two
parent strands

single stranded DNA binding proteins

attach to the unwound strands preventing
them from winding back together.
 A primase repeatedly synthesises
RNA primers about 10 nucleotides
long on the lagging strand to initiate
synthesis of Okazaki fragments by
DNA polymerase.

RNA primer
Assembly of DNA
polymerase on the
lagging strand
DNA synthesis on lagging strand
A nuclease deletes the RNA primers between completed
Okazaki fragments
A ligase seals the final nick
between the two ends of the
new DNA fragments on the
lagging strand.
 Telomeres

lagging template strand

Nowhere to put
next RNA primer
RNA
primer

telomerase adds additional

repeats to the template strand

DNA synthesis from RNA primer

DNA repair
completes lagging strand
23
 Quiz; Draw the replication bubble, below, and,

A. Indicate where the origin of replication was located (use O).

B. Label the leading-strand template and the lagging-strand
template of the right-hand fork [R] as X and Y, respectively.
C. Indicate by arrows the direction in which the newly made DNA
strands (indicated by dark lines) were synthesized.
D. Number the Okazaki fragments on each strand 1, 2, and 3 in
the order in which they were synthesized.
E. Indicate where the most recent DNA synthesis has occurred
(use S).
F. Indicate the direction of movement of the replication forks with
arrows. 24
 Quiz; Draw the replication bubble, below, and,

A. Indicate where the origin of replication was located (use O).

B. Label the leading-strand template and the lagging-strand
template of the right-hand fork [R] as X and Y, respectively.
C. Indicate by arrows the direction in which the newly made DNA
strands (indicated by dark lines) were synthesized.
D. Number the Okazaki fragments on each strand 1, 2, and 3 in
the order in which they were synthesized.
E. Indicate where the most recent DNA synthesis has occurred
(use S).
F. Indicate the direction of movement of the replication forks with
arrows. 25
 DNA Repair
Changes in DNA sequence can arise from

mistakes in copying

damage from the environment

These mistakes could become lethal if they are not rapidly repaired.
Failure leads to a permanent change called a mutation.

e.g. the disease sickle-cell anemia is caused by a single A to T base

change (mutation) in one of the 500 bases of DNA that code for the
protein haemoglobin.
 DNA Repair

The error rate in replication is 1 in 107 but 109 bases are copied
every time one of our cells divides. Errors in replication produce
mismatches e.g. A paired with G. 99% of these errors are
immediately corrected by mismatch repair enzymes that operate
exclusively on the newly synthesised strand.
DNA polymerase; separate active sites for
DNA synthesis and editing.
Mismatch Repair
Mismatch Repair

Incorrectly changing the

template strand results in
two mutant daughter
duplexes.
Mismatch Repair

Correctly recognising and

repairing the newly
synthesised strand
corrects the error.
35
 Depurination
NH 2

N
N

N
N
HO

A H
H H

H
OH H

H2O

NH 2
HO
OH
N
O N
H H

H H N
OH H H N

abasic site
 NH2
Deamidation
N

N O

HO

C H
O
H

H H
OH H

H2O
O

NH

N O

HO

U H
O
H
+ NH3

H H
OH H
Ultraviolet light causes DNA damage by
forming thymine dimers.
Damaged bases are usually
different from the standard
bases and can be recognised
 Recombination
Recombination is an important process in
evolution, allowing organisms to change in
response to changes in environment.

In homologous recombination, two double

stranded DNA molecules that have regions of
identical sequence exchange strands.
Breaking and resealing the strands leads to
exchange of DNA between the two
molecules.
Double-stranded break
This structure is a
Holliday Junction

Exchange of genetic
material between
chromosomes A & B
Resolving a Holliday junction to give two
DNA duplexes.

resolvase
resolvase
rotation

Recombination

No recombination