3.

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
Mismatched repair
Excision repair
iii). Defects in DNA repair or replication
 The mammalian cell cycle

Rapid growth and DNA synthesis and

preparation for histone synthesis
DNA synthesis
S
phase

G0 G1
phase
Quiescent cells
G2
phase Growth and
M preparation for
phase
cell division

Mitosis
DNA replication is semi-conservative

Parental DNA strands

Each of the parental strands serves as a

template for a daughter strand

Daughter DNA strands

Origins of DNA replication on mammalian chromosomes
origins of DNA replication (every ~150 kb)

5’ 3’
3’ 5’

bidirectional replication

replication bubble

fusion of bubbles
5’ 3’
3’ 5’
daughter chromosomes
5’ 3’
3’ 5’
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
 DNA DNA

Reaction catalyzed by DNA polymerase

• 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
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
 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 the 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

• 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
 Properties of DNA polymerases
DNA polymerases of humans
a b g d e
Location nucl nucl mito nucl nucl
Replication yes no yes yes (no)
Repair no yes no no yes3
Functions
5’ to 3’ polymerase yes yes yes yes yes
3’ to 5’ exonuclease no no yes yes yes
5’ to 3’ exonuclease1 no no no no no
Primase yes no no no no
Associates with PCNA2 no no no yes no
Processivity low high
Strand synthesis lagging repair both leading repair

1 activitypresent in associated proteins

2 Proliferating Cell Nuclear Antigen
3involved in transcription-linked DNA repair
 Proteins at the replication fork in humans

leading strand
helicase
PCNA 3’
pol d 5’

5’
3’
DNA ligase

5’ to 3’ exo
SSB associated
with the
pol a
complex
pol e
topoisomerases I and II
primase activity
lagging strand associated with pol a
 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).
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

New germline mutations

• each sperm contains ~100 new mutations

• a normal ejaculate has ~100 million sperm
• 100 X 100 million = 10 billion new mutations
• ~1 in 10 sperm carries a new deleterious mutation
• at a rate of production of ~8 X 107 sperm per day,
a male will produce a sperm with a new mutation
in the Duchenne muscular dystrophy gene
approximately every 10 seconds.
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 and C G
• replication of C-G should give daughter strands each with C-G

C G C A and C G
• 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

Derivation by hydroxylamine

Alkylation by dimethyl sulfate causes depurination

The formation of a quarternary nitrogen destabilizes the

deoxyriboside bond and the base is released from deoxyribose
Attack by oxygen radicals
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 formationSpontaneous 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 mismatched (post-replication) repair followed by
excision repair

• Mutations that occur spontaneously any time are repaired by

excision repair (base excision or nucleotide excision)
 Mismatched (post-replication) repair

• 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 (base or nucleotide)
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 b DNA polymerase b
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
DNA ligase DNA ligase
ATGCCGCATTGA ATGCCGCATTGATAG
TACGGCGTAACT TACGGCGTAACTATC
Base excision repair Nucleotide excision repair
 Deamination of cytosine can be repaired

Deamination of 5-methylcytosine cannot be repaired

More than 30% of all single base changes that have been detected
as a cause of genetic disease have occurred at 5’-mCG-3’ sites
Defects in DNA repair or replication
• Xeroderma pigmentosum
• Ataxia telangiectasia
• Fanconi anemia
• Bloom syndrome 100
• Cockayne syndrome human
elephant

cow
Life span

10

Correlation between DNA repair hamster

activity in fibroblast cells from rat
various mammalian species and mouse
the life span of the organism 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 leukemia
• Xeroderma pigmentosum
• caused by mutations in genes involved in nucleotide excision repair
• associated with a 2000-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
• increased risk of X-ray
• 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