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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 mutationii). Spontaneous mutations in DNA replication
iii). Lesions caused by mutagens
c). DNA repair
i). Types of lesions that require repair
ii). Mechanisms of repairProofreading by DNA polymerase
Mismatch repair
Excision repair
iii). Defects in DNA repair or replication
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Themammalian cell cycle
G1
S
G2
M
G0
DNA synthesis andhistone synthesis
Growth andpreparation for
cell division
Rapid growth andpreparation for
DNA synthesis
Quiescent cells
phase
phase
phase
phase
Mitosis
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DNA replication is semi-conservative
Parental DNA strands
Daughter DNA strands
Each of the parental strands serves as a
template for a daughter strand
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origins of DNA replication (every ~150 kb)
replication bubble
daughter chromosomes
fusion of bubbles
bidirectional replication
Origins of DNA replication on mammalian chromosomes
53
35
5
3
3
5
3
5
5
3
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Initiation of DNA synthesis at the E. coli origin (ori)
5
3
3
5
origin DNA sequence
binding of dnaA proteins
A A A
dnaA proteins coalesce
DNA melting induced
by the dnaA proteinsA
A
A
AA
A
A
A
A
AA
A
B C
dnaB and dnaC proteins bind
to the single-stranded DNA
dnaB further unwinds the helix
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AA
A
AA
A B C
dnaB further unwinds the helix
and displaces dnaA proteins
G
dnaG (primase) binds...
A
A
A
A A
AB C
G
...and synthesizes an RNA primer
RNA primer
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B C
G
5 3template strand
RNA primer
(~5 nucleotides)
Primasome
dna B (helicase)
dna C
dna G (primase)
OH3 5
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3
5 3
RNA primer
newly synthesized DNA
5
5
DNA polymerase
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Discontinuous synthesis of DNA
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.
5
3
3
5
5
3
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3
5
5 3
3 5
5
3
3
5
5
3
leading strand (synthesized continuously)
lagging strand (synthesized discontinuously)
Each replication fork has a leading and a lagging strand
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 calledOkazaki fragments (100-1000 bases in length)
replication fork replication fork
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RNA primer
53
3
5
3
5
direction of leading strand synthesis
direction of lagging strand synthesis
replication fork
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53
3
5
3
5
Strand separation at the replication fork causes positive
supercoiling of the downstream double helix
DNA gyrase is a topoisomerase II, whichbreaks 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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5
3 5
3
Movement of the replication fork
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Movement of the replication fork
RNA primer
Okazaki fragmentRNA primer
5
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3
RNA primer
5
DNA polymerase III initiates at the primer and
elongates DNA up to the next RNA primer
5
5
3
5
newly synthesized DNA (100-1000 bases)
(Okazaki fragment)
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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
pol III
pol I
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newly synthesized DNA
(Okazaki fragment)5
3
53
DNA ligase seals the gap by catalyzing the formationof a 3, 5-phosphodiester bond in an ATP-dependent reaction
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5
3
3
5
Proteins at the replication fork in E. coli
Rep protein (helicase)
Single-strand
binding protein
(SSB)
BCG Primasome
pol I
pol III
pol III
DNA ligase
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 aheadof 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 gapDNA 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 primerwith 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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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
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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
T f b i t ti
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Types of base pair mutations
CATTCACCTGTACCA
GTAAGTGGACATGGT
CATGCACCTGTACCA
GTACGTGGACATGGT
CATCCACCTGTACCA
GTAGGTGGACATGGT
transition (T-A to C-G) transversion (T-A to G-C)
CATCACCTGTACCA
GTAGTGGACATGGT
deletion
CATGTCACCTGTACCA
GTACAGTGGACATGGT
insertion
base pair substitutions
transition: pyrimidine to pyrimidine
transversion: pyrimidine to purine
normal sequence
deletions and insertions can involve oneor more base pairs
S t t ti b d b t t
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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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Guanine
Thymine
KETO ENOL
Tautomeric forms of the DNA bases
Mutation caused by tautomer of cytosine
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Mutation caused by tautomer of cytosine
Cytosine
Cytosine
Guanine
Adenine
cytosine mispairs with adenine resulting in a transition mutation
Normal tautomeric form
Rare imino tautomeric form
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Mutation is perpetuated by replication
replication of C-G should give daughter strands each with C-G
tautomer formation Cduring replication will result in mispairingand 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
C G C G
C G C A
AC T A
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Chemical mutagens
Deamination by nitrous acid
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N
NH
NH
N
NH2
O
N
NH
NH
NH
NH2
O
O
Attack by oxygen free radicals
leading to oxidative damage
guanine
8-oxyguanine (8-oxyG)
many different oxidative modifications occur by smoking, etc. 8-oxyG causes G to T transversions
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
S f DNA l i
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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 hypoxanthineDeletion-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
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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)
Mi t h ( t li ti ) i
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Mismatch (post-replication) repair(reduces DNA replication errors 1,000-fold)
5
3
CH3
CH3
CH3
CH3
the parental DNA strands are
methylated on certainadenine bases
mutations on the newly
replicated strand are
identified by scanningfor mismatches prior to
methylation of the newly
replicated DNA
the mutations are repaired
by excision repair mechanisms after repair, the newly
replicated strand is methylated
E i i i
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Excision repair
ATGCUGCATTGATAG
TACGGCGTAACTATC
thymine dimer
AT AGTACGGCGTAACTATC
ATGCCGCATTGATAG
TACGGCGTAACTATC
ATGCCGCATTGATAG
TACGGCGTAACTATC
excinuclease
DNA polymerase
DNA ligase
(~30 nucleotides)
ATGCUGCATTGA
TACGGCGTAACT
ATGCGCATTGA
TACGGCGTAACT
AT GCATTGATACGGCGTAACT
deamination
ATGCCGCATTGA
TACGGCGTAACT
ATGCCGCATTGA
TACGGCGTAACT
uracil DNA glycosylase
repair nucleases
DNA polymerase
DNA ligase
Base excision repair Nucleotide excision repair
D i ti f t i b i d
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Deamination of cytosine can be repaired
More than 30% of all single base changes that have been detectedas a cause of genetic disease have occurred at 5-mCpG-3 sites
Deamination of 5-methylcytosine cannot be repaired
cytosine uracil
thymine5-methyl-
cytosine
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DNA repair activity
Life
span
1
10
100human
elephant
cow
hamsterratmouseshrew
Correlation between DNA repair
activity in fibroblast cells from
various mammalian species and
the life span of the organism
Defects in DNA repair or replication
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Defects in DNA repair or replicationAll 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