3 lectures: DNA Replication,

Mutation, Repair
 Learning Objectives for Lecture 2:

• Understand the general mechanism of DNA replication

• Understand the need for a primer for DNA replication
• Understand the dynamics of DNA strand synthesis on the leading and lagging
strands of the replication fork
• Understand the general functions of the proteins involved in DNA replication
at the replication fork
• Understand the role of DNA gyrase in DNA replication and know the class of
antibiotics that inhibits this enzyme
• Understand the types of mutations and the rates with which DNA is mutated
• Understand the mechanism by which mutations are generated
• Understand the basic types of DNA repair and when they take place
• Understand the reason for 5'-mCpG mutation hot spots in DNA
• Understand the significance of high fidelity DNA replication and for the
presence of DNA repair mechanisms for cellular function and for human
disease
 2. DNA Replication, Mutation, Repair

a). DNA replication

i). Cell cycle/ semi-conservative replication
ii). Initiation of DNA replication
iii). Discontinuous DNA synthesis
iv). Components of the replication apparatus
b). Mutation
i). Types and rates of mutation
ii). Spontaneous mutations in DNA replication
iii). Lesions caused by mutagens
c). DNA repair
i). Types of lesions that require repair
ii). Mechanisms of repair
Proofreading by DNA polymerase
Mismatch repair
Excision repair
iii). Defects in DNA repair or replication
 Section 1

General Concepts of
DNA Replication
 DNA replication
• A reaction in which daughter DNAs are
synthesized using the parental DNAs as the
template.
• Transferring the genetic information to the
descendant generation with a high fidelity

replicatio
n

parental DNA
daughter DNA
 Daughter strand synthesis
• Chemical formulation:

• The nature of DNA replication is a

series of 3´- 5´phosphodiester bond
formation catalyzed by a group of
enzymes.
Phosphodiester bond formation
 DNA replication system
Template: double stranded DNA
Substrate: dNTP
Primer: short RNA fragment with a
free 3´-OH end
Enzyme: DNA-dependent DNA
polymerase (DDDP),
other enzymes,
protein factor
Characteristics of replication

 Semi-conservative replication
 Bidirectional replication
 Semi-continuous replication
 High fidelity
§1.1 Semi-Conservative Replication
 Semiconservative replication

Half of the parental DNA molecule is

conserved in each new double helix,
paired with a newly synthesized
complementary strand. This is called
semiconservative replication
Semiconservative replication
Experiment of DNA semiconservative replication

"Heavy" DNA(15N)

grow in 14N
medium

The first
generation

grow in 14N
medium

The second
generation
 Significance

The genetic information is ensured to be

transferred from one generation to the
next generation with a high fidelity.
§1.2 Bidirectional Replication

• Replication starts from unwinding the

dsDNA at a particular point (called
origin), followed by the synthesis on
each strand.
• The parental dsDNA and two newly
formed dsDNA form a Y-shape
structure called replication fork.
 Replication fork

5'
3'

3' 5'
3'
5'

5'

direction of 3'

replication
 Bidirectional replication

• Once the dsDNA is opened at the

origin, two replication forks are
formed spontaneously.
• These two replication forks move in
opposite directions as the syntheses
continue.
Bidirectional replication
Replication of prokaryotes

The replication
process starts
from the
origin, and
proceeds in
two opposite
directions. It
is named θ
replication.
 Replication of eukaryotes

• Chromosomes of eukaryotes have

multiple origins.
• The space between two adjacent
origins is called the replicon, a
functional unit of replication.
origins of DNA replication (every ~150 kb)
§1.3 Semi-continuous Replication

The daughter strands on two template

strands are synthesized differently since
the replication process obeys the
principle that DNA is synthesized from
the 5´ end to the 3´end.
 Leading strand

On the template having the 3´- end, the

daughter strand is synthesized
continuously in the 5’-3’ direction. This
strand is referred to as the leading
strand.

3'
5' 3' 5'
direction of unwinding
3'
5'
Semi-continuous replication
3'

5'
replication fork

3'
5'
3'
replication direction 5'

3'

5'
Okazaki fragment

3'

5'
leading strand
 Okazaki fragments
• Many DNA fragments are synthesized
sequentially on the DNA template
strand having the 5´- end. These DNA
fragments are called Okazaki
fragments. They are 1000 – 2000 nt
long for prokaryotes and 100-150 nt
long for eukaryotes.
• The daughter strand consisting of
Okazaki fragments is called the
lagging strand.
 Semi-continuous replication

Continuous synthesis of the leading

strand and discontinuous synthesis of
the lagging strand represent a unique
feature of DNA replication. It is
referred to as the semi-continuous
replication.
 Section 2

Enzymology
of DNA Replication
 Enzymes and protein factors
protein Mr # function

Dna A protein 50,000 1 recognize origin

Dna B protein 300,000 6 open dsDNA
Dna C protein 29,000 1 assist Dna B binding
DNA pol Elongate the DNA strands

Dna G protein 60,000 1 synthesize RNA primer

SSB 75,600 4 single-strand binding

DNA topoisomerase 400,000 4 release supercoil

constraint
§2.1 DNA Polymerase
DNA-pol of prokaryotes
• The first DNA-
dependent DNA
polymerase (short for
DNA-pol I) was
discovered in 1958 by
Arthur Kornberg who
received Nobel Prize in
physiology or medicine
in 1959.
• Later, DNA-pol II and DNA-pol III
were identified in experiments using
mutated E.coli cell line.
• All of them possess the following
biological activity.
1. 5′ →3′ polymerizing
2. exonuclease
DNA-pol of E. coli
 DNA-pol I

• Mainly
responsible for
proofreading
and filling the
gaps, repairing
DNA damage
 Klenow fragment
N end DNA-pol Ⅰ C end

caroid

• small fragment (323 AA): having 5´→3´

exonuclease activity
• large fragment (604 AA): called Klenow
fragment, having DNA polymerization
and 3´→5´exonuclease activity
 DNA-pol II

• Temporary functional when DNA-pol I

and DNA-pol III are not functional
• Still capable for doing synthesis on
the damaged template
• Participating in DNA repairing
 DNA-pol III
• A heterodimer enzyme composed of
ten different subunits
• Having the highest polymerization
activity (105 nt/min)
• The true enzyme responsible for the
elongation process
 Structure of DNA-pol III

α ： has 5´→ 3´
polymerizing activity
ε ： has 3´→ 5´
exonuclease activity
and plays a key role to
ensure the replication
fidelity.
θ: maintain
heterodimer structure
 DNA-pol of eukaryotes

DNA-pol α : initiate replication DnaG,

and synthesize primers primase
DNA-pol β : replication with repairing
low fidelity

DNA-pol γ : polymerization in
mitochondria
DNA-pol δ : elongation DNA-pol
III
DNA-pol ε : proofreading and DNA-pol I
filling gap
§2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers
using free NTPs as the substrate and
the ssDNA as the template.
• Primers are short RNA fragments of a
several decades of nucleotides long.
• Primers provide free 3´-OH groups to
react with the α -P atom of dNTP to
form phosphoester bonds.
• Primase, DnaB, DnaC and an origin
form a primosome complex at the
initiation phase.
§2.3 Helicase

• Also referred to as DnaB.

• It opens the double strand DNA with
consuming ATP.
• The opening process with the
assistance of DnaA and DnaC
§2.4 SSB protein
• Stand for single strand DNA binding
protein
• SSB protein maintains the DNA
template in the single strand form in
order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
§2.5 Topoisomerase

• Opening the dsDNA will create

supercoil ahead of replication forks.
• The supercoil constraint needs to be
released by topoisomerases.
• The interconversion of topoisomers
of dsDNA is catalyzed by a
topoisomerase in a three-step
process:
• Cleavage of one or both strands
of DNA
• Passage of a segment of DNA
through this break
• Resealing of the DNA break
 Topoisomerase I (topo I)

• Also called ω -protein in prokaryotes.

• It cuts a phosphoester bond on one
DNA strand, rotates the broken DNA
freely around the other strand to relax
the constraint, and reseals the cut.
 Topoisomerase II (topo II)
• It is named gyrase in prokaryotes.
• It cuts phosphoester bonds on both
strands of dsDNA, releases the
supercoil constraint, and reforms the
phosphoester bonds.
• It can change dsDNA into the
negative supercoil state with
consumption of ATP.
§2.6 DNA Ligase

3' 5'
5' 3'
RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
• Connect two adjacent ssDNA strands
by joining the 3´-OH of one DNA
strand to the 5´-P of another DNA
strand.
• Sealing the nick in the process of
replication, repairing, recombination,
and splicing.
§2.7 Replication Fidelity
• Replication based on the principle of
base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use two mechanisms to
ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
 Proofreading and correction
• DNA-pol I has the function to correct
the mismatched nucleotides.
• It identifies the mismatched
nucleotide, removes it using the 3´- 5´
exonuclease activity, add a correct
base, and continues the replication.
 Exonuclease functions
5´→3´ 3´→5´
exonuclease exonuclease
activity activity
cut primer or excise mismatched
excise mutated nuleotides
segment
5' 3'
C T T C A G G A

G A A G T C C G G C G
3' 5'
 Section 3

DNA Replication
Process
 Sequential actions

• Initiation: recognize the starting point,

separate dsDNA, primer synthesis, …
• Elongation: add dNTPs to the existing
strand, form phosphoester bonds,
correct the mismatch bases, extending
the DNA strand, …
• Termination: stop the replication
§3.1 Replication of prokaryotes
a. Initiation

• The replication starts at a particular

point called origin.
• The origin of E. coli, ori C, is at the
location of 82.
• The structure of the origin is 248 bp
long and AT-rich.
Genome of E. coli
 Structure of ori C

• Three 13 bp consensus sequences

• Two pairs of anti-consensus repeats
Formation of preprimosome
 Formation of replication fork
• DnaA recognizes ori C.
• DnaB and DnaC join the DNA-DnaA
complex, open the local AT-rich
region, and move on the template
downstream further to separate
enough space.
• DnaA is replaced gradually.
• SSB protein binds the complex to
stabilize ssDNA.
 Primer synthesis

• Primase joins and forms a complex

called primosome.
• Primase starts the synthesis of
primers on the ssDNA template using
NTP as the substrates in the 5´- 3´
direction at the expense of ATP.
• The short RNA fragments provide free
3´-OH groups for DNA elongation.
 Releasing supercoil constraint
• The supercoil constraints are
generated ahead of the replication
forks.
• Topoisomerase binds to the dsDNA
region just before the replication
forks to release the supercoil
constraint.
• The negatively supercoiled DNA
serves as a better template than the
positively supercoiled DNA.
 Primosome complex

Dna B Dna C
Dna A primase 3'

5'
3'

5'
DNA topomerase
b. Elongation

• dNTPs are continuously connected to

the primer or the nascent DNA chain
by DNA-pol III.
• The core enzymes (α 、、 and θ )
catalyze the synthesis of leading and
lagging strands, respectively.
• The nature of the chain elongation is
the series formation of the
phosphodiester bonds.
• The synthesis
direction of the
leading strand is
the same as that of
the replication fork.

• The synthesis
direction of the
latest Okazaki
fragment is also the
same as that of the
replication fork.
 Lagging strand synthesis
• Primers on Okazaki fragments are
digested by RNase.
• The gaps are filled by DNA-pol I in the
5´→3´direction.
• The nick between the 5´end of one
fragment and the 3´end of the next
fragment is sealed by ligase.
3' 5'
5' 3'

RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
c. Termination

• The replication of E. coli is

bidirectional from one origin, and the
two replication forks must meet at
one point called ter at 32.
• All the primers will be removed, and
all the fragments will be connected
by DNA-pol I and ligase.
§3.2 Replication of Eukaryotes
• DNA replication is closely related
with cell cycle.
• Multiple origins on one
chromosome, and replications are
activated in a sequential order rather
than simultaneously.
Cell cycle
 Initiation

• The eukaryotic origins are shorter

than that of E. coli.
• Requires DNA-pol α (primase
activity) and DNA-pol δ (polymerase
activity and helicase activity).
• Needs topoisomerase and
replication factors (RF) to assist.
b. Elongation

• DNA replication and nucleosome

assembling occur simultaneously.
• Overall replication speed is
compatible with that of prokaryotes.
c. Termination
3' 5'
5' 3'
3' 5'
5' 3'

connection of discontinuous
segment
3' 5'
5' 3'
3' 5'
5' 3'
 Telomere
• The terminal structure of eukaryotic
DNA of chromosomes is called
telomere.
• Telomere is composed of terminal
DNA sequence and protein.
• The sequence of typical telomeres is
rich in T and G.
• The telomere structure is crucial to
keep the termini of chromosomes in
the cell from becoming entangled and
sticking to each other.
 Telomerase
• The eukaryotic cells use telomerase to
maintain the integrity of DNA telomere.
• The telomerase is composed of
telomerase RNA
telomerase association protein
telomerase reverse transcriptase

• It is able to synthesize DNA using RNA

as the template.
VEDIO….TELOMERASE
 Significance of Telomerase

• Telomerase may play important

roles is cancer cell biology and in
cell aging.
Initiation of DNA synthesis at the E. coli origin (ori)
origin DNA sequence
5’ 3’
3’ A A A 5’
binding of dnaA proteins

A
A A
DNA melting induced
A A A by the dnaA proteins

dnaA proteins coalesce

dnaB and dnaC proteins bind
to the single-stranded DNA
A
A A
A A A B C
dnaB further unwinds the helix
 dnaG (primase) binds...

A
A A G
A A B C
A
dnaB further unwinds the helix
and displaces dnaA proteins

A ...and synthesizes an RNA primer

A
A G
A B C RNA primer
A
A
 Primasome
G dna B (helicase)
B C dna C
dna G (primase)

template strand
5’ 3’
3’ OH 5’
RNA primer
(~5 nucleotides)
 DNA polymerase

5’ 3’
5’
RNA primer

3’
5’
newly synthesized DNA
Discontinuous synthesis of DNA

5’ 3’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
3’ 5’

Because DNA is always synthesized in a 5’ to 3’ direction,

synthesis of one of the strands...

5’
3’
...has to be discontinuous.

This is the lagging strand.

Each replication fork has a leading and a lagging strand

leading strand (synthesized continuously)

replication fork replication fork

5’ 3’
5’ 3’ 5’ 3’
3’ 5’ 3’ 5’
3’ 5’

lagging strand (synthesized discontinuously)

• The leading and lagging strand arrows show the direction

of DNA chain elongation in a 5’ to 3’ direction
• The small DNA pieces on the lagging strand are called
Okazaki fragments (100-1000 bases in length)
RNA primer
direction of leading strand synthesis
3’
5’
replication fork
5’
3’

3’
5’
direction of lagging strand synthesis
 Strand separation at the replication fork causes positive
supercoiling of the downstream double helix

3’
5’

5’
3’

3’
• DNA gyrase is a topoisomerase II, which 5’
breaks and reseals the DNA to introduce negative
supercoils ahead of the fork
• Fluoroquinolone antibiotics target DNA gyrases in many
gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)
 Movement of the replication fork

5’
3’ 5’

3’
Movement of the replication fork

5’ RNA primer
Okazaki fragment
RNA primer
 RNA primer pol III
5’ 5’
3’

DNA polymerase III initiates at the primer and

elongates DNA up to the next RNA primer

5’ 5’
3’

newly synthesized DNA (100-1000 bases) pol I

(Okazaki fragment)
5’
3’

DNA polymerase I inititates at the end of the Okazaki fragment

and further elongates the DNA chain while simultaneously
removing the RNA primer with its 5’ to 3’ exonuclease activity
 newly synthesized DNA
(Okazaki fragment)
5’
3’

DNA ligase seals the gap by catalyzing the formation

of a 3’, 5’-phosphodiester bond in an ATP-dependent reaction
5’
3’
 Proteins at the replication fork in E. coli

Rep protein (helicase)

3’
pol III
5’

5’
3’ G Primasome DNA ligase
C B
Single-strand
binding protein
(SSB)
pol III

DNA gyrase - this is a topoisomerase II, which

breaks and reseals double-stranded DNA to introduce
negative supercoils ahead of the fork
pol I
 Components of the replication apparatus

dnaA binds to origin DNA sequence

Primasome
dnaB helicase (unwinds DNA at origin)
dnaC binds dnaB
dnaG primase (synthesizes RNA primer)
DNA gyrase introduces negative supercoils ahead
of the replication fork
Rep protein helicase (unwinds DNA at fork)
SSB binds to single-stranded DNA
DNA pol III primary replicating polymerase
DNA pol I removes primer and fills gap
DNA ligase seals gap by forming 3’, 5’-phosphodiester bond
 Properties of DNA polymerases

DNA polymerases of E. coli_

pol I pol II pol III (core)

Polymerization: 5’ to 3’ yes yes yes
Proofreading exonuclease: 3’ to 5’ yes yes yes
Repair exonuclease: 5’ to 3’ yes no no

DNA polymerase III is the main replicating enzyme

DNA polymerase I has a role in replication to fill gaps and excise
primers on the lagging strand, and it is also a repair enzyme
and is used in making recombinant DNA molecules

• all DNA polymerases require a primer with a free 3’ OH group

• all DNA polymerases catalyze chain growth in a 5’ to 3’ direction
• some DNA polymerases have a 3’ to 5’ proofreading activity
 Mutation

Types and rates of mutation

Type Mechanism Frequency________

Genome chromosome 10-2 per cell division
mutation missegregation
(e.g., aneuploidy)

Chromosome chromosome 6 X 10-4 per cell division

mutation rearrangement
(e.g., translocation)

Gene base pair mutation 10-10 per base pair per

mutation (e.g., point mutation, cell division or
or small deletion or 10-5 - 10-6 per locus per
insertion generation
 Mutation rates* of selected genes

Gene New mutations per 106 gametes

Achondroplasia 6 to 40
Aniridia 2.5 to 5
Duchenne muscular dystrophy 43 to 105
Hemophilia A 32 to 57
Hemophilia B 2 to 3
Neurofibromatosis -1 44 to 100
Polycystic kidney disease 60 to 120
Retinoblastoma 5 to 12

*mutation rates (mutations / locus / generation) can vary

from 10-4 to 10-7 depending on gene size and whether
there are “hot spots” for mutation (the frequency at most
loci is 10-5 to 10-6 ).
 Many polymorphisms exist in the genome

• the number of existing polymorphisms is ~1 per 500 bp

• there are ~5.8 million differences per haploid genome
• polymorphisms were caused by mutations over time
• polymorphisms called single nucleotide polymorphisms
(or SNPs) are being catalogued by the Human
Genome Project as an ongoing project
Types of base pair mutations
normal sequence
CATTCACCTGTACCA
GTAAGTGGACATGGT
transition (T-A to C-G) transversion (T-A to G-C)
CATCCACCTGTACCA CATGCACCTGTACCA
GTAGGTGGACATGGT GTACGTGGACATGGT
base pair substitutions
transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine

deletion insertion
CATCACCTGTACCA CATGTCACCTGTACCA
GTAGTGGACATGGT GTACAGTGGACATGGT
deletions and insertions can involve one
or more base pairs
Spontaneous mutations can be caused by tautomers
Tautomeric forms of the DNA bases

Adenine

Cytosine

AMINO IMINO
 Tautomeric forms of the DNA bases

Guanine

Thymine

KETO ENOL
 Mutation caused by tautomer of cytosine

Cytosine

Normal tautomeric form Guanine

Cytosine

Rare imino tautomeric form Adenine

• cytosine mispairs with adenine resulting in a transition mutation
 Mutation is perpetuated by replication

C G C G
• replication of C-G should give daughter strands each with C-G

C G C A
• tautomer formation C during replication will result in mispairing
and insertion of an improper A in one of the daughter strands

C A T A
• which could result in a C-G to T-A transition mutation in the next
round of replication, or if improperly repaired
 Chemical mutagens

Deamination by nitrous acid

 Attack by oxygen free radicals O
leading to oxidative damage
N
NH

• many different oxidative modifications occur NH N NH2

• by smoking, etc.
• 8-oxyG causes G to T transversions guanine
O
H
N
NH
O
NH N NH2
8-oxyguanine (8-oxyG)

• the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation

• mutation of the MTH1 gene causes increased tumor formation in mice
 Ames test for mutagen detection

• named for Bruce Ames

• reversion of histidine mutations by test compounds
• His- Salmonella typhimurium cannot grow without histidine
• if test compound is mutagenic, reversion to His+ may occur
• reversion is correlated with carcinogenicity
Thymine dimer formation by UV light
 Summary of DNA lesions
Missing base Acid and heat depurination (~104 purines
per day per cell in humans)

Altered base Ionizing radiation; alkylating agents

Incorrect base Spontaneous deaminations
cytosine to uracil
adenine to hypoxanthine

Deletion-insertion Intercalating reagents (acridines)

Dimer formation UV irradiation

Strand breaks Ionizing radiation; chemicals (bleomycin)

Interstrand cross-links Psoralen derivatives; mitomycin C

Tautomer formation Spontaneous and transient
 Mechanisms of Repair

• Mutations that occur during DNA replication are repaired when

possible by proofreading by the DNA polymerases

• Mutations that are not repaired by proofreading are repaired

by mismatch (post-replication) repair followed by
excision repair

• Mutations that occur spontaneously any time are repaired by

excision repair (base excision or nucleotide excision)
 Mismatch (post-replication) repair
(reduces DNA replication errors 1,000-fold)

• the parental DNA strands are

methylated on certain CH3
adenine bases
CH3
• mutations on the newly
replicated strand are
5’ identified by scanning
3’ for mismatches prior to
CH3
methylation of the newly
replicated DNA

• the mutations are repaired

by excision repair mechanisms
• after repair, the newly CH3
replicated strand is methylated
 Excision repair
deamination
ATGCUGCATTGA
TACGGCGTAACT
uracil DNA glycosylase thymine dimer
ATGC GCATTGA ATGCUGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
repair nucleases excinuclease
AT GCATTGA AT (~30 nucleotides) AG
TACGGCGTAACT TACGGCGTAACTATC
DNA polymerase β DNA polymerase β
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
DNA ligase DNA ligase
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
Base excision repair Nucleotide excision repair
 Deamination of cytosine can be repaired

cytosine uracil

Deamination of 5-methylcytosine cannot be repaired

5’-methyl- thymine
cytosine

More than 30% of all single base changes that have been detected
as a cause of genetic disease have occurred at 5’-mCpG-3’ sites
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism

100
human
elephant

cow
Life span

10

hamster
rat
mouse
shrew
1
DNA repair activity
Defects in DNA repair or replication
All are associated with a high frequency of chromosome
and gene (base pair) mutations; most are also associated with a
predisposition to cancer, particularly leukemias
• Xeroderma pigmentosum
• caused by mutations in genes involved in nucleotide excision repair
• associated with a >1000-fold increase of sunlight-induced
skin cancer and with other types of cancer such as melanoma
• Ataxia telangiectasia
• caused by gene that detects DNA damage
• increased risk of X-ray
• associated with increased breast cancer in carriers
• Fanconi anemia
• caused by a gene involved in DNA repair
• increased risk of X-ray and sensitivity to sunlight
• Bloom syndrome
• caused by mutations in a a DNA helicase gene
• increased risk of X-ray
• sensitivity to sunlight
• Cockayne syndrome
• caused by a defect in transcription-linked DNA repair
• sensitivity to sunlight
• Werner’s syndrome
• caused by mutations in a DNA helicase gene
• premature aging