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DNA Replication, Mutation, and 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

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
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