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
DNA synthesis and
histone synthesis
Rapid growth and
preparation for
DNA synthesis
S
phase
G0
Quiescent cells
G1
phase
G2
M
phase
Mitosis
phase
Growth and
preparation for
cell division
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’
5’
3’
daughter chromosomes
3’
5’
3’
5’
Initiation of DNA synthesis at the E. coli origin (ori)
origin DNA sequence
5’
3’
A
A
3’
5’
A
binding of dnaA proteins
A
A
A
A
DNA melting induced
by the dnaA proteins
A
A
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
B C
A
A
dnaB further unwinds the helix
and displaces dnaA proteins
A
A
A
A
A
A
B C
...and synthesizes an RNA primer
G
RNA primer
G
B C
5’
Primasome
dna B (helicase)
dna C
dna G (primase)
template strand
3’ OH
5’
RNA primer
(~5 nucleotides)
3’
DNA polymerase
5’
5’
RNA primer
5’
newly synthesized DNA
3’
3’
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
3’
5’
5’
3’
5’ 3’
3’ 5’
5’
3’
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.
3’
5’
Each replication fork has a leading and a lagging strand
leading strand (synthesized continuously)
replication fork
5’
3’
3’
5’
replication fork
5’ 3’
3’ 5’
5’
3’
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
3’
RNA primer
5’
pol III
5’
DNA polymerase III initiates at the primer and
elongates DNA up to the next RNA primer
3’
3’
5’
newly synthesized DNA (100-1000 bases)
(Okazaki fragment)
5’
5’
pol I
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
3’
5’
newly synthesized DNA
(Okazaki fragment)
DNA ligase seals the gap by catalyzing the formation
of a 3’, 5’-phosphodiester bond in an ATP-dependent reaction
3’
5’
Proteins at the replication fork in E. coli
Rep protein (helicase)
3’
5’
pol III
5’
3’
G
Primasome
DNA ligase
C B
Single-strand
binding protein
(SSB)
DNA gyrase - this is a topoisomerase II, which
breaks and reseals the DNA to introduce
negative supercoils ahead of the fork
pol III
pol I
Components of the replication apparatus
dnaA
Primasome
dnaB
dnaC
dnaG
DNA gyrase
Rep protein
SSB
DNA pol III
DNA pol I
DNA ligase
binds to origin DNA sequence
helicase (unwinds DNA at origin)
binds dnaB
primase (synthesizes RNA primer)
introduces negative supercoils ahead
of the replication fork
helicase (unwinds DNA at fork)
binds to single-stranded DNA
primary replicating polymerase
removes primer and fills gap
seals gap by forming 3’, 5’-phosphodiester bond
Properties of DNA polymerases
DNA polymerases of E. coli_
Polymerization: 5’ to 3’
Proofreading exonuclease: 3’ to 5’
Repair exonuclease: 5’ to 3’
pol I pol II pol III (core)
yes yes yes
yes yes yes
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
a
DNA polymerases of humans
b
g
d
Location
nucl nucl mito nucl
Replication
yes
no
yes
yes
Repair
no
yes
no
yes
Functions
5’ to 3’ polymerase
yes
yes
yes
yes
3’ to 5’ exonuclease
no
no
yes
yes
5’ to 3’ exonuclease1
no
no
no
no
Primase
yes
no
no
no
Associates with PCNA2
no
no
no
yes
Processivity
low
high
Strand synthesis
lagging* repair both leading*
* see notes below
1 activity present in associated proteins
2 Proliferating Cell Nuclear Antigen – “sliding clamp”
3 involved in transcription-linked DNA repair
e
nucl
yes
yes3
yes
yes
no
no
yes
lagging*
Proteins at the replication fork in humans
helicase
leading strand
PCNA
pol d
5’
3’
3’
5’
DNA ligase
SSB
topoisomerases I and II
5’ to 3’ exo
associated
with the
complex
pol e
pol a
(or pol d)
primase activity
lagging strand associated with pol a
Mutation
Types and rates of mutation
Type
Genome
mutation
Mechanism
chromosome
missegregation
(e.g., aneuploidy)
Frequency________
10-2 per cell division
Chromosome
mutation
chromosome
rearrangement
(e.g., translocation)
6 X 10-4 per cell division
Gene
mutation
base pair mutation
(e.g., point mutation,
or small deletion or
insertion
10-10 per base pair per
cell division or
10-5 - 10-6 per locus per
generation
Mutation rates* of selected genes
Gene
New mutations per 106 gametes
Achondroplasia
Aniridia
Duchenne muscular dystrophy
Hemophilia A
Hemophilia B
Neurofibromatosis -1
Polycystic kidney disease
Retinoblastoma
6
2.5
43
32
2
44
60
5
to 40
to
5
to 105
to 57
to
3
to 100
to 120
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)
CATCCACCTGTACCA
GTAGGTGGACATGGT
transversion (T-A to G-C)
CATGCACCTGTACCA
GTACGTGGACATGGT
base pair substitutions
transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine
deletion
CATCACCTGTACCA
GTAGTGGACATGGT
insertion
CATGTCACCTGTACCA
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
adenine bases
CH3
5’
3’
CH3
• the mutations are repaired
by excision repair mechanisms
• after repair, the newly
replicated strand is methylated
CH3
• mutations on the newly
replicated strand are
identified by scanning
for mismatches prior to
methylation of the newly
replicated DNA
CH3
Excision repair (base or nucleotide)
deamination
ATGCUGCATTGA
TACGGCGTAACT
uracil DNA glycosylase
ATGC GCATTGA
TACGGCGTAACT
repair nucleases
AT
GCATTGA
TACGGCGTAACT
DNA polymerase b
ATGCCGCATTGA
TACGGCGTAACT
DNA ligase
thymine dimer
ATGCUGCATTGATAG
TACGGCGTAACTATC
excinuclease
AT (~30 nucleotides) AG
TACGGCGTAACTATC
DNA polymerase b
ATGCCGCATTGATAG
TACGGCGTAACTATC
DNA ligase
ATGCCGCATTGA
TACGGCGTAACT
ATGCCGCATTGATAG
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
human
• Cockayne syndrome
elephant
Life span
cow
10
hamster
rat
mouse
shrew
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism
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