Lectures 5 and 6

DNA Replication
The Structure of DNA Provides a Mechanism for Heredity
 Arthur Kornberg – DNA replication in vitro
worked with E. coli cells – goal was to study DNA replication
in vitro – in a test tube.
Step one – Develop an in vitro assay for DNA synthesis
• grind up E. coli cells to obtain a cell extract that contains all cellular
proteins (including the unknown enzyme that makes DNA)
• add 14C-Thymine deoxynucleotide (14C-dTTP)+ all 4 dNTPs + DNA
• incubate for a few hours to allow DNA synthesis to occur
• add perchloric acid  stops reaction and precipitates DNA
• small amount of 14C-dTTP goes from perchloric acid soluble to
insoluble (incorporated into DNA?).
• Add DNase enzyme  14C-dTTP goes back into soluble fraction.
Something in the crude cell extract is synthesizing DNA
 Arthur Kornberg – DNA replication in vitro

Step 2 – purify the DNA synthesizing “activity” from the

crude protein fraction.
• involves separating crude protein into “fractions” and testing each
fraction for DNA synthesizing ability
 This purified DNA polymerase can be used to study the protein
components of DNA Polymerase and to study the mechanism
of DNA replication
How does DNA synthesis proceed?
Model 1: random nucleotide addition

5’ 3’
Model 2: 5’ to 3’ synthesis

5’
5’ 3’
Model 3: 3’ to 5’ synthesis

3’
5’ 3’
Kornberg’s evidence that DNA synthesis is 5’ to 3’

14
C-dTTP

3’to5’’ exonuclease

5’ 3’
5’
3’

pulse of 14C-dTTP
pulse of 3’to5’ exonuclease
 radioactivity goes back to perchloric acid soluble fraction.
If DNA synthesis was 3’ to 5’

14
C-dTTP

3’to5’ exonuclease

3’ 5’
3’
5’

pulse of 14C-dTTP
pulse of 3’to5’ exonuclease
 radioactivity would remain in perchloric acid insoluble fraction
Kornberg’s evidence that DNA synthesis is 5’ to 3’

• start DNA synthesis in presence of “cold” nucleotides

• short pulse of radioactive nucleotide to label only most
recently added nucleotides.
• add 3’to5’ exonuclease  radioactivity is lost from DNA
(becomes acid soluble)
• add 5’to3’ exonuclease  radioactivity is retained on DNA
• Therefore nucleotides are added to the 3’ end of a DNA
molecule.
DNA synthesis results in release of pyrophasphate
 Kornberg’s evidence that DNA synthesis involves
release of pyrophosphate from deoxynucleotide
triphosphates (dNTPs)

Experiment: template + primer + 4 dNTPs + 14C-dATP-P32

deoxyribose
Result: Release of 32P pyrophosphate at equal molarity to
amount of 14C-nucleoside incorporated into DNA.
Base-Pairing Underlies DNA Replication and DNA Repair
 3’
5’

5’
3’

3’
5’

continuous (3’ to 5’) discontinuous (5’ to 3’)

 DNA REPLICATION MECHANISMS
• The DNA Replication Fork Is Asymmetrical

• Leading strand – 5’ to 3’ synthesis in direction of

fork movement
• How is the lagging strand synthesized?
– studies by Reiji and Tsuneko Okazaki - 1968
Evidence for lagging strand synthesis

The experiment:

• brief pulse of H3-Thymidine

to label DNA
• alkali denaturation
• sucrose gradient to separate
DNA according to size
• measure radioactivity in each
fraction

Okazaki, 1968
Evidence for lagging strand synthesis

Okazaki, 1968
Evidence for lagging strand synthesis

*
*

*
*
Short pulse –5-10 sec

Okazaki, 1968
Evidence for lagging strand synthesis

*
*

*
*

*
*

*
*

*
*

*
*

*
*
*
*
*
*
Longer pulse – 60 sec

Okazaki, 1968
How are the Okazaki fragments linked together?

Discovery of DNA ligase – enzyme that ligates 5’ and 3’ ends

together
Temperature sensitive mutants for E. coli DNA ligase gene –
lethal at restrictive temperature
If ligase is responsible for linking Okazaki fragments –
predict:

Short pulse: *
*
How are the Okazaki fragments linked together?

Discovery of DNA ligase – enzyme that ligates 5’ and 3’ ends

together
Temperature sensitive mutants for E. coli ligase gene – lethal
at restrictive temperature
If ligase is responsible for linking Okazaki fragments –
predict:

*
*
Long pulse: *
*
*
*
Lagging strand synthesis depends on DNA ligase

restrictive temp permissive temp

The DNA Replication Fork Is Asymmetrical

Lagging strand synthesis

 Lagging strand synthesis
Experiments by Okazaki
• showed that newly synthesized DNA on the
lagging strand occurs in short discontinuous
fragments that soon become linked to older DNA –
Okazaki fragments
• the short DNA fragments are linked by DNA ligase
• the lagging strand is primed by a short RNA
molecule
 Lagging strand synthesis
DNA Primase
• synthesizes a short RNA primer on lagging strand
DNA Primase Synthesizes Short RNA Primer Molecules on the
Lagging Strand
DNA Primase Synthesizes Short RNA Primer Molecules on the
Lagging Strand
 Lagging strand synthesis
Experiments by Okazaki
• the short DNA fragments also contain RNA
– add RNAse to reaction
– add H3-Uridine instead of H3-dT
DNA Primase Synthesizes Short RNA Primer Molecules on the
Lagging Strand
THE INITIATION AND COMPLETION OF
DNA REPLICATION IN CHROMOSOMES
• Prokaryote genomes typically contain a single well
characterized replication origin

• Eukaryotic Chromosomes Contain Multiple Origins

of Replication
– In yeast, Origins are well characterized
specific sequences
– In most eukaryotes, origins are not well
defined.
DNA Synthesis Begins at Replication Origins
Major components of DNA replication
machinery
• DNA helicase (Mcm) opens up the double helix
• Single stranded DNA binding protein (RPA)
stabilizes ssDNA (particularly important on lagging
strand)
• Sliding clamp (PCNA) – encircles DNA and keeps
DNA polymerase from falling off
• DNA polymerase
 DNA REPLICATION MECHANISM
• DNA helicase opens Up the DNA Double Helix in
Front of the Replication Fork
DNA Helicase Open Up the DNA Double Helix in Front of the
Replication Fork
Single-stranded DNA-binding protein (RPA)
PCNA - A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA
 DNA REPLICATION MECHANISM
• Primase puts down small RNA primer  DNA/RNA
hybrid
• PCNA ring encircles DNA/RNA hybrid.
• PCNA helps bring DNA Polymerase to DNA
• PCNA stabilizes DNA pol association with DNA
– Leading strand – PCNA and DNA pol remain
bound – continuous synthesis
– Lagging strand – PCNA and DNA pol fall off
when they encounter 5’ end of previous
Okazaki fragment
The Proteins at a Replication Fork Cooperate to Form a Replication
Machine
The Proteins at a Replication Fork Cooperate to Form a Replication
Machine
 DNA REPLICATION MECHANISMS
• The Proteins at a Replication Fork Cooperate to
Form a Replication Machine
• The replication fork contains a single large
complex including polymerases for leading and
lagging strand

• Lagging strand is looped around so that its

polymerase can associate with leading strand
polymerase
THE COMPLETION OF DNA REPLICATION
IN CHROMOSOMES

Sheltrin

• DNA synthesis on the lagging strand is incomplete

• Ends of chromosomes (telomeres) contain repeat
sequences
• These repeats are bound by Sheltrin complex
– Protects chromosome ends
– Attracts Telomerase  extension of ends
Telomerase Replicates the Ends of Chromosomes
Telomerase Replicates the Ends of Chromosomes

Sheltrin

Sheltrin
 Telomeres
• Telomeres Are Protected by telomere binding
proteins – sheltrin complex
• Sheltrin recruits Telomerase
• Telomerase uses bound RNA as template for
extending 3’ end of DNA
• Primase inserts a primer near 3’ end
• DNA polymerase extends this
 DNA and RNA Polymerases
• DNA-dependent DNA polymerases – DNA
replication
• RNA-dependent DNA polymerases – Telomerase,
Reverse transcriptase from retroviruses
 DNA and RNA Polymerases
• DNA-dependent DNA polymerases – DNA
replication
• RNA-dependent DNA polymerases – Telomerase,
Reverse transcriptase from retroviruses
• DNA-dependent RNA polymerases – transcription
• RNA-dependent RNA polymerases – eg. Influenza,
Sars-Cov-2
 Three Mechanisms That Ensure
Accuracy of DNA Replication
– base pairing precedes covalent attachment
of nucleotide
– 3’ to 5’ exonuclease activity
– mismatch repair
Polymerase and exonuclease activity are found in distinct parts of
DNA polymerase
 3’ to 5’ Exonuclease Activity
If a mismatched nucleotide is incorporated,
Exonuclease activity removes mismatched
nucleotide

Exonuclease activity resides in a distinct domain or

subunit of DNA Polymerase

Exonuclease function first identified in bacteriophage

T7 - mutations in DNA polymerase gene that
produce a “mutator” phenotype
 Three Mechanisms That Ensure
Accuracy of DNA Replication
– base pairing precedes covalent attachment
of nucleotide
– 3’ to 5’ exonuclease activity
– mismatch repair
A Strand-Directed Mismatch Repair System Removes Replication
Errors That Escape from the Replication Machine

MSH2 MLH1
 DNA Mismatch Repair
• A Strand-Directed Mismatch Repair System
Removes Replication Errors That Escape from the
Replication Machine

– MSH2 and MLH1 protein complex scans newly

synthesized DNA (they are part of the large
replication machine at each replication fork)
– detects mismatches
The Proteins at a Replication Fork Cooperate to Form a Replication
Machine
 DNA Mismatch Repair
• A Strand-Directed Mismatch Repair System
Removes Replication Errors That Escape from the
Replication Machine

– MSH2 and MLH1 protein complex scans newly

synthesized DNA
– detects mismatches
– Makes nicks in “new” DNA strand on either side
of mismatch
– strand removal and repair by DNA pol I

– families with mutations in these genes have

very high cancer incidence
Lecture 7

DNA Repair
Genetic Screens Identified radiation sensitive yeast mutants

rad mutant

mutant cells proliferate on control plate

rad mutant

replica plate grown in

presence of low level UV
radiation

rad mutants – unable to survive low level UV radiation

- also unable to survive other mutagens at levels that
wild type cells can tolerate
 DNA REPAIR
• Identification of genes required for DNA repair –
Rad genes – from large scale mutagenesis of
haploid yeast
– plate out single cells
– grow colonies
– replica plate
– low level UV or other radiation added to replica
plate
– isolate colonies that grow on untreated but not
on rad treated plates  rad mutants

Most yeast rad genes encode proteins required for

DNA repair.
Same genes are often tumour suppressors in humans
Introduction
 DNA DAMAGE
• Two general categories of DNA damage with respect to
effect on the DNA:
– Chemical alteration of a nucleotide
• Failure to repair may result in a change in DNA
sequence at the next S-phase. Eg. deamination,
depurination
• Or may result in a failure of DNA polymerase (and
RNA polymerase) to progress past the altered
nucleotide. Eg. Pyrimidine dimers
– Break in the DNA backbone – either a single stranded
or a double stranded break.
• Failure to repair will result in failure of DNA
polymerase (and RNA polymerase) to progress
past the break.
 DNA DAMAGE
• Many of these forms of DNA damage occur
spontaneously in all cells
• Specific DNA damaging agents can greatly
increase the frequency of specific types of DNA
damage
– Eg. UV light  increase pyrimidine dimers
– Nitrous acid  increase cytosine
deamination
– X-rays  increase double stranded breaks
Examples of Sites of Spontaneous DNA Damage that Lead to Change
in DNA Sequences
Without DNA Repair, Spontaneous DNA Damage Would Rapidly
Change DNA Sequences
Without DNA Repair, Spontaneous DNA Damage Would Rapidly
Change DNA Sequences
Without DNA Repair, Spontaneous DNA Damage Would Rapidly
Change DNA Sequences
 DNA REPAIR

– Repair of DNA damage can be carried out by

error-free pathways or by error-prone pathways
– Use of error-prone pathways leads to mutations and
can lead to cancer.
 DNA REPAIR
• DNA Damage Can Be Removed by More Than One
Pathway

• Detection of an altered or missing base

– Base Excision Repair (BER) – altered base
– Nucleotide Excision Repair (NER) – pyrimidine
dimers
– Translesion “Repair” – error prone
BER for repair of altered bases:

1. Scanning of double helix to

detect altered bases
2. Glycosylases specific to each
type of altered base

3. AP Endonuclease +
Phosphodiesterase
4. DNA Polymerase, ligase

Repair of depurinated DNA:

last steps of BER pathway
Pyrimidine dimers (Thymine/Cytosine dimers)
Nucleotide excision repair and Xeroderma Pigmentosum
Responses to pyrimidine dimers

Nucleotide excision repair – error-free pathway

If NER pathway fails, DNA synthesis cannot

proceed.
 Translesion repair – error-prone pathway
 Nucleotide Excision Repair
• In humans, this pathway is critical for repair of
Pyrimidine dimers caused by UV-irradiation

• Xeroderma Pigmentosa – disease in which

individuals are extremely sensitive to sunlight
– several different genes: XPA,XPB etc (most also
identified as Rad genes in yeast)
– high cancer rate
– some (eg. XPG) also associated with neural
disorders, reduced growth, premature aging
XPC-Rad23 dimer
scans DNA, recognizes
damage.

Recruit other repair

proteins – excision of
damaged strand – XPG
(excision nuclease)

Repair via DNA

polymerase, ligase
Error-prone Translesion DNA Polymerases Are Used in Emergencies
Eg. Pyrimidine dimer
 Translesion DNA polymerase
• if DNA damage is not repaired (by NER or other
pathway) DNA polymerase stalls
• exchange of DNA pol for a translesion DNA
polymerase
• This polymerase will insert “best guess”
nucleotides to allow bypass of lesion
• Error-prone pathway
• Only used if other pathways cannot cope
• In XP individuals this pathway is highly active 
high mutation rate  cancer
 Transcription Coupled Repair (TCR)
• mammalian cells accumulate thymine dimers
more outside genes than within genes
– therefore DNA that is transcribed is less likely to be
mutated

– OR repair of DNA damage occurs more efficiently in

transcribed genes
• failure to repair DNA damage in coding genes
results in stalling of RNA polymerase
• Perhaps there’s a DNA repair pathway that
specifically detects damage in transcribed genes
 Cockayne Syndrome

– UV sensitivity syndrome
• like XP though not as severe. Most UV-
induced Thymine dimers can still be repaired

– growth defects, neurological disorders,

premature aging – all due to high level of cell
death.
• These phenotypes are also seen in some XP
syndromes (eg. XPG) but not others (eg.
XPC)
 Cockayne Syndrome (CS)

– Venema (1990)
• Grew cells from CS patient in vitro
• CS cells have same frequency of mutations
in genes compared to non-coding DNA
• failure to repair DNA damage in coding
genes results in stalling of RNA polymerase
• cell lethality due to stalled transcription

– Therefore the gene that is altered in Cockayne

syndrome is necessary for transcription coupled
DNA repair
Transcription coupled repair
Transcription
stalled at DNA
damage
RNA polymerase II
encounters DNA damage
CSB
and stalls

CSB (Cockayne
Syndrome B) identifies
stalled RNA Polymerase

CSB recruits repair

factors (eg. XPG –
excision nuclease)

Repair via DNA

polymerase, ligase
 DNA REPAIR
• Coupling Nucleotide Excision Repair to
Transcription Ensures That the Cell’s Most
Important DNA Is Efficiently Repaired
• Also ensures that transcription can proceed
through damaged DNA
XPC-Rad23 dimer
scans DNA, recognizes
damage.

Recruit other repair

proteins – excision of
damaged strand – XPG
(excision nuclease)

Repair via DNA

polymerase, ligase
 Nucleotide Excision Repair
• Important for repair of UV-induced Thymine dimers
• Identification of DNA damage
• Excision of damaged base(s) and several on either
side
• Repair via repair DNA polymerase
• Two branches:
– general repair pathway identifies DNA damage
anywhere in genome
– TCR pathway – stalled transcription (due to DNA
damage) triggers repair
– some genes function in both branches, some in
only one
 DNA REPAIR
• Double-Strand Breaks Repaired via two major
pathways
• Non-homologous end joining (NHEJ)
– error prone
– ends are simply joined – ends are recessed
before joining– results in small deletion
• Homologous recombination (HR)
– accurate – uses sister chromatid as template for
repair
Double-Strand Breaks Are Efficiently Repaired
 HOMOLOGOUS RECOMBINATION
• Homologous Recombination Can Flawlessly Repair
Double-Strand Breaks in DNA

• Sister chromatid serves as template for repair of

the break
NHEJ Pathway

Ku binding to DNA ends

 Processing of ends (some

loss of nucleotides)
 Ligation
Using Cas9/CRISPR system for gene editing

Cas9 mediates a ds break in genomic DNA

 NHEJ pathway repairs it
Using CRISPR to generate small deletions)

A) introduce Cas9 plus a single guide RNA corresponding

to a site within gene  small deletion
Using CRISPR to introduce specific point mutations in a gene

Waaijers, 2014

-plasmid sequence contains gene sequence that has

been altered by site directed mutagenesis

-useful for testing significance of specific amino

acids, putative domains
Using CRISPR to introduce an epitope tag

Waaijers, 2014

- plasmid contains tag sequence + ~1kb of flanking

sequence on either side
introduce plasmid + gRNA + Cas9
 ds break near one end of coding sequence
 repair via homologous recombination