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

The mammalian cell cycle

DNA synthesis and
histone synthesis

Rapid growth and

preparation for
DNA synthesis

phase

G0
Quiescent cells

G1

phase

G2

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

3
5

binding of dnaA proteins

A
A

DNA melting induced

by the dnaA proteins

dnaA proteins coalesce

dnaB and dnaC proteins bind
to the single-stranded DNA

A
A

A
A

B C

dnaB further unwinds the helix

dnaG (primase) binds...

A
A

B C

A
A

dnaB further unwinds the helix

and displaces dnaA proteins

A
A
A

A
A

B C

...and synthesizes an RNA primer

G
RNA primer

G
B C

Primasome
dna B (helicase)
dna C
dna G (primase)

template strand
3 OH
5
RNA primer
(~5 nucleotides)

DNA polymerase
5

5
RNA primer

5
newly synthesized DNA

Discontinuous synthesis of DNA

5
3

3
5

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

Strand separation at the replication fork causes positive

supercoiling of the downstream double helix
3
5
5
3
3
5

DNA gyrase is a topoisomerase II, which

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

RNA primer
Okazaki fragment
RNA primer

RNA primer
5

pol III
5
DNA polymerase III initiates at the primer and
elongates DNA up to the next RNA primer

newly synthesized DNA (100-1000 bases)

(Okazaki fragment)
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

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

Proteins at the replication fork in E. coli

Rep protein (helicase)

3
5

pol III

5
3

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
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
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
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
Achondroplasia
Aniridia
Duchenne muscular dystrophy
Hemophilia A
Hemophilia B
Neurofibromatosis -1
Polycystic kidney disease
Retinoblastoma

New mutations per 10 6 gametes

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).

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)

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

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

leading to oxidative damage

many different oxidative modifications occur

by smoking, etc.
8-oxyG causes G to T transversions

NH

NH
N

NH2

guanine

O
H
N

NH

O
NH

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
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

deamination

Excision repair

ATGCUGCATTGA
TACGGCGTAACT
uracil DNA glycosylase

thymine dimer

ATGC GCATTGA
TACGGCGTAACT

ATGCUGCATTGATAG
TACGGCGTAACTATC

AT
GCATTGA
TACGGCGTAACT

AT (~30 nucleotides) AG
TACGGCGTAACTATC

ATGCCGCATTGA
TACGGCGTAACT

ATGCCGCATTGATAG
TACGGCGTAACTATC

ATGCCGCATTGA
TACGGCGTAACT

ATGCCGCATTGATAG
TACGGCGTAACTATC

Base excision repair

Nucleotide excision repair

repair nucleases

DNA polymerase

DNA ligase

excinuclease

DNA polymerase

DNA ligase

Deamination of cytosine can be repaired

cytosine

uracil

Deamination of 5-methylcytosine cannot be repaired

5-methylcytosine

thymine

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

Life span

cow
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
Werners syndrome
caused by mutations in a DNA helicase gene
premature aging