DNA: Replication

Replication Facts

• DNA has to be copied before a cell

divides
• DNA is copied during the S or synthesis
phase of interphase
• New cells will need identical DNA
strands

2
 Synthesis Phase (S phase)
• S phase during interphase of the cell cycle
• Nucleus of eukaryotes

S
DNA replication phase
takes
place in the S phase.
G1 interphase G2

Mitosis
-prophase
-metaphase
-anaphase
-telophase 3
 DNA Replication
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
– conservative model
– semiconservative model
– dispersive model

4
5
 DNA Replication
Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.

Each strand of DNA acts as a template for

the synthesis of a new strand.

8
 DNA Replication
• DNA replication is the process of copying a DNA molecule. Replication is
semiconservative, with each strand of the original double helix (parental molecule)
serving as a template (mold or model) for a new strand in a daughter molecule. This
process consists of:
• Unwinding (initiation): old strands of the parent DNA molecule are unwound as
weak hydrogen bonds between the paired bases are “unzipped” and broken by the
enzyme helicase.
• Complementary base pairing (elongation): free nucleotides present in the nucleus
bind with complementary bases on unzipped portions of the two strands of DNA; this
process is catalyzed by DNA polymerase.
• Joining (elongation): complementary nucleotides bond to each other to form new
strands; each daughter DNA molecule contains an old strand and a new strand; this
process is also catalyzed by DNA polymerase.
• Termination – replication is terminated differently in prokaryotes and eukaryotes
Ends in prokaryotes when origin is reached
Ends in eukaryotes when telomere is reached
telomeres – repeated DNA sequence on the ends of eukaryotic
chromosomes

• DNA replication must occur before a cell can divide; in cancer, drugs with molecules
similar to the four nucleotides are used to stop replication. 9
10
 Modes of Replication

• Replicons: units of replication

• Replication origin

• Theta replication: circular DNA, E. coli; single

origin of replication forming a replication fork,
usually a bidirectional replication

• Rolling-circle replication: virus, F factor of

E. coli; single origin of replication
 Linear Eukaryotic Replication

• Eukaryotic cells; thousands of origins; a typical

replicon: 200,000 ~ 300,000 bp in length
 Initiation
How is a Repl. origin selected?

Priming at the oriC (Bacterial)

Origin
+ ATP
+ Hu on the origin

Ready to bind primase!

Order of events at OriC

1. Many copies of dnaA bind the four 9-mers; DNA wraps

around dnaA forming “Initial Complex”. This requires ATP
and a protein Hu that is already bound to the DNA.

2. This triggers opening of the 13-mers (Open complex).

3. Two copies of dnaB (helicase) bind the 13-mers. This

requires dnaC (which does not remain with the
Prepriming Complex) and ATP.

4. Primase binds to dnaB (helicase) and the DNA.

5. dnaB:primase complex moves along the template 3’>5’

synthesizing RNA primers 5’>3’ for Pol III to extend.
Replication Elongation
 Enzymes Involved in
Elongation:

1. DNA-dependent DNA polymerases

– synthesize DNA from dNTPs
– require a template strand and a primer strand with
a 3’-OH end
– all synthesize from 5’ to 3’ (add nt to 3’ end only)
Comparison of E.coli DNA Polymerases I and III

1 subunit

10 subunits
22
 Proofreading Activity

Insertion of the wrong nucleotide causes the DNA

polymerase to stall, and then the 3’-to-5’ exonuclease
activity removes the mispaired A nt. The polymerase then
continues adding nts to the primer.
If DNA polymerases only synthesize 5’ to 3’, how
does the replication fork move directionally?
• Lagging strand synthesized as small (~100-1000 bp)
fragments - “Okazaki fragments” .

• Okazaki fragments begin as very short 6-15 nt RNA

primers synthesized by primase.

2. Primase - RNA polymerase that synthesizes the

RNA primers (11-12 nt that start with pppAG) for both
lagging and leading strand synthesis
26
Lagging strand synthesis (continued)

Pol III extends the RNA primers until the 3’ end of an

Okazaki fragment reaches the 5’ end of a downstream
Okazaki fragment.

Then, Pol I degrades the RNA part with its 5’-3’

exonuclease activity, and replaces it with DNA. Pol I
is not highly processive, so stops before going far.
At this stage, Lagging strand is a series of DNA
fragments (without gaps).

Fragments stitched together covalently by DNA

Ligase.

3. DNA Ligase - joins the 5’ phosphate of one DNA

molecule to the 3’ OH of another, using energy in the
form of NAD (prokaryotes) or ATP (eukaryotes). It
prefers substrates that are double-stranded, with only
one strand needing ligation, and lacking gaps.
 DNA Ligase Substrate Specificity

Ligase will join these two G--G--A--T--C--C--T--T--G--A--T--C--C

| | | | | | | | | | | | |
C--C--T--A--G G--A--A--C--T--A--G--G

Ligase will NOT join these G--G--A--T--C--C--T--T--G--A--T--C--C

two. | | | | | | | | | | | |
C--C--T--A--G C--A--A--C--T--A--G--G

Ligase will NOT join these G--G--A--T--C--C--T--T--G--A--T--C--C

two. | | | | | | | | | | | |
C--C--T--A--A G--A--A--C--T--A--G--G

Ligase will NOT join these G--G--A--T--C--C--T--T--G--A--T--C--C

two. | | | | | | | | | | | |
C--C--T--A--G G--T--A--C--T--A--G--G

Ligase will NOT join these

two. C--C--T--A--G C--T--A--C--T--A--G--G
Mechanism of
Prokaryotic Ligase

DNA Ligase-
P
P HO
5' 3' NAD NMN
+AMP

Ligase binds NAD, 1

cleaves it, leaving AMP NMN 3' P
attached to it.
Ligase-AMP binds and Ligase
1
attaches to 5’ end of a
DNA molecule (1) via the
+
NAD
AMP 3'
P AMP
(Eukaryotic
2
AMP. DNA ligase
HO uses ATP
The DNA fragment with as AMP
donor,
the 3’ OH end (2) reacts
with the phosphodiester,
AMP
+ instead of
displacing the AMP- NAD).
ligase.
1 2
P
3' 5'
Other proteins needed for DNA replication:

4. DNA Helicase (dnaB gene) – hexameric protein,

unwinds DNA strands, uses ATP.

5. SSB – single-strand DNA binding protein, prevents

strands from re-annealing and from being degraded,
stimulates DNA Pol III.

6. Gyrase – Topoisomerase II, keeps DNA ahead of

fork from over winding (i.e., relieves torsional strain).

Replisome - DNA and protein machinery at a

replication fork.
DNA Supercoiling
 Rubber Band Model
of Supercoiling DNA

DNA Gyrase relaxes positive

supercoils by breaking and
rejoining both DNA strands.
 A Replisome
5' Primase Primosome
3'

Helicase (DnaB)
Gyrase
DNA Polymerase I extends 5'
one Okazaki fragment and
removes the RNA from
another. Sp inn ing
3'
DNA Ligase then joins at 10,000
fragments together. 5'
rp m

DNA Polymerase III

acts here

ssDNA binding
protein (SSB)
5' A prokaryotic fork is travelling at 50 to 100 kb / m inute.

3' Eukaryotic forks travel at 0.5 - 5 kb / m inute.

What about the ends (or telomeres) of linear chromosomes?

DNA polymerase/ligase cannot fill gap at end of chromosome after RNA

primer is removed. this gap is not filled, chromosomes would
become shorter each round of replication!

Solution:

1. Eukaryotes have tandemly repeated sequences at the ends of their

chromosomes.

2. Telomerase (composed of protein and RNA complementary to the

telomere repeat) binds to the terminal telomere repeat and
catalyzes the addition of of new repeats.

3. Compensates by lengthening the chromosome.

4. Absence or mutation of telomerase activity results in chromosome

shortening and limited cell division.
Fig. 3.16 Synthesis of telomeric DNA by telomerase

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Final Step - Assembly into Nucleosomes:

• As DNA unwinds, nucleosomes must disassemble.

• Histones and the associated chromatin proteins must be duplicated

by new protein synthesis.

• Newly replicated DNA is assembled into nucleosomes almost

immediately.

• Histone chaperone proteins control the assembly.

Fig. 3.17
 Proofreading New DNA

• DNA polymerase initially makes about 1 in

10,000 base pairing errors
• Enzymes proofread and correct these
mistakes
• The new error rate for DNA that has been
proofread is 1 in 1 billion base pairing
errors

38
Prokaryotic Versus Eukaryotic Replication

39
• Prokaryotic Replication
• Bacteria have a single loop of DNA that must
replicate before the cell divides.
• Replication in prokaryotes may be bidirectional
from one point of origin or in only one direction.
• Replication only proceeds in one direction, from 5'
to 3'.
• Bacterial cells are able to replicate their DNA at a
rate of about 106 base pairs per minute.
• Bacterial cells can complete DNA replication in 40
minutes; eukaryotes take hours.
• Eukaryotic Replication
• Replication in eukaryotes starts at many
points of origin and spreads with many
replication bubbles—places where the DNA
strands are separating and replication is
occurring.
• Replication forks are the V-shape ends of
the replication bubbles; the sites of DNA
replication.
• Eukaryotes replicate their DNA at a slower
rate – 500 to 5,000 base pairs per minute.
• Eukaryotes take hours to complete DNA
replication.
41
42
43
44
• Replication Errors
• A genetic mutation is a permanent change in the sequence
of bases.
• Base changes during replication are one way mutations
occur.
• A mismatched nucleotide may occur once per 100,000 base
pairs, causing a pause in replication.
• Proofreading is the removal of a mismatched nucleotide;
DNA repair enzymes perform this proofreading function
and reduce the error rate to one per billion base pairs.
• Incorrect base pairs that survive the proofreading process
contribute to gene mutations.

45
Model of DNA replication