2' DNA Replication, Mutation, Repair

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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 Mism
atch repair Excision repair iii). Defects
in DNA repair or replication
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The mammalian cell cycle
DNA synthesis and histone synthesis
Rapid growth and preparation for DNA
synthesis
S
phase
G1
G0
phase
Quiescent cells
G2
phase
Growth and preparation for cell division
M
phase
Mitosis
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DNA replication is semi-conservative
Parental DNA strands
Each of the parental strands serves as a
template for a daughter strand
Daughter DNA strands
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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
3 5
5 3
5
Initiation of DNA synthesis at the E. coli origin
(ori)
origin DNA sequence
binding of dnaA proteins
DNA melting induced by the dnaA proteins
dnaA proteins coalesce
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dnaG (primase) binds...
G
B
dnaB further unwinds the helix and displaces
dnaA proteins
...and synthesizes an RNA primer
G
B
RNA primer
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Primasome dna B (helicase) dna C
dna G (primase)
template strand
5
3
OH
3
5
RNA primer (5 nucleotides)
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DNA polymerase
5
3
5
RNA primer
3
5
newly synthesized DNA
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Discontinuous synthesis of DNA
5 3
3 5
5 3
3 5
5 3
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.
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Each replication fork has a leading and a lagging
strand
leading strand (synthesized continuously)
replication fork
replication fork
5 3
3 5
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)

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RNA primer
direction of leading strand synthesis
3 5
replication fork
5 3
3 5
direction of lagging strand synthesis
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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)

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Movement of the replication fork
5 3
5
3
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Movement of the replication fork
RNA primer
5
Okazaki fragment
RNA primer
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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)
(Okazaki fragment)
pol I
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
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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
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Proteins at the replication fork in E. coli
Rep protein (helicase)
3 5
pol III
5 3
Primasome
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
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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
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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

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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
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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).
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• 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

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Types of base pair mutations
normal sequence
CATTCACCTGTACCA GTAAGTGGACATGGT
transition (T-A to C-G)
transversion (T-A to G-C)
CATGCACCTGTACCA GTACGTGGACATGGT
CATCCACCTGTACCA GTAGGTGGACATGGT
base pair substitutions transition pyrimidine
to pyrimidine transversion pyrimidine to
purine
deletion
insertion
CATCACCTGTACCA GTAGTGGACATGGT
CATGTCACCTGTACCA GTACAGTGGACATGGT
deletions and insertions can involve one
or more base pairs
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Spontaneous mutations can be caused by tautomers
Tautomeric forms of the DNA bases
Adenine
Cytosine
AMINO
IMINO
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Tautomeric forms of the DNA bases
Guanine
Thymine
KETO
ENOL
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Mutation caused by tautomer of cytosine
Cytosine
Guanine
Normal tautomeric form
Cytosine
Adenine
Rare imino tautomeric form

• cytosine mispairs with adenine resulting in a
  transition mutation

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Mutation is perpetuated by replication

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

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

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

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Chemical mutagens
Deamination by nitrous acid
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O
Attack by oxygen free radicals leading to
oxidative damage
N
N
H

• many different oxidative modifications occur
• by smoking, etc.
• 8-oxyG causes G to T transversions

N
H
N
N
H
2
guanine
O
H
N
N
H
O
N
H
N
N
H
2
8-oxyguanine (8-oxyG)

• the MTH1 protein degrades 8-oxy-dGTP preventing
  misincorporation
• mutation of the MTH1 gene causes increased tumor
  formation in mice

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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

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Thymine dimer formation by UV light
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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 Intercalati
ng reagents (acridines) Dimer formation UV
irradiation Strand breaks Ionizing radiation
chemicals (bleomycin) Interstrand
cross-links Psoralen derivatives mitomycin C
Tautomer formation Spontaneous and transient
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• 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)

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Mismatch (post-replication) repair (reduces DNA
replication errors 1,000-fold)

• the parental DNA strands are
• methylated on certain
• adenine bases

• mutations on the newly
• replicated strand are
• identified by scanning
• for mismatches prior to
• methylation of the newly
• replicated DNA

5 3

• the mutations are repaired
• by excision repair mechanisms
• after repair, the newly
• replicated strand is methylated

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Excision repair
thymine dimer
ATGCUGCATTGATAG TACGGCGTAACTATC
excinuclease
AT AG TACGGCGTAACTATC
(30 nucleotides)
DNA polymerase b
ATGCCGCATTGATAG TACGGCGTAACTATC
DNA ligase
ATGCCGCATTGATAG TACGGCGTAACTATC
Base excision repair
Nucleotide excision repair
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Deamination of cytosine can be repaired
cytosine
Deamination of 5-methylcytosine cannot be repaired
thymine
5-methyl- 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
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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
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• 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 gt1000-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