dna mutation 3

8/13/2019 Dna Mutation 3

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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 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

Mismatched repair

Excision repair

iii). Defects in DNA repair or replication


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The mammalian 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

5’ 3’

3’ 5’

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 helixand 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’ 3’ template 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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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

DNA DNA


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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

5’ 3’

3’

5’

3’

5’

direction of leading strand synthesis

direction of lagging strand synthesis

replication fork


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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 andelongates DNA up to the next RNA primer

5’

5’

3’

5’

newly synthesized DNA (100-1000 bases)

(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

pol III

pol I


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newly synthesized DNA

(Okazaki fragment)5’

3’

5’ 3’

DNA ligase seals the gap by catalyzing the formationof a 3’, 5’-phosphodiester bond in an ATP-dependent reaction

Proteins at the replication fork in E coli


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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 the 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

• 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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DNA polymerases of humansb g d eLocation nucl nucl mito nucl nuclReplication yes no yes yes (no)

Repair no yes no no yes3

Functions

5’ to 3’ polymerase yes yes yes yes yes3’ to 5’ exonuclease no no yes yes yes

5’ to 3’ exonuclease1 no no no no no

Primase yes no no no no

Associates with PCNA2 no no no yes no

Processivity low highStrand synthesis lagging repair both leading repair

1 activity present in associated proteins

2 Proliferating Cell Nuclear Antigen3involved in transcription-linked DNA repair

Proteins at the replication fork in humans


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5’

3’

3’ 5’

Proteins at the replication fork in humans

helicase

SSB

pol

DNA ligase

topoisomerases I and II

PCNA

primase activity

associated with pol a

pol dleading strand

lagging strand

5’ to 3’ exo

associated

with thecomplex

pol e

Mutation


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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 permutation (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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Polymorphisms exist in the genome


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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


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ypes o base pa utat o s

CATTCACCTGTACCA

GTAAGTGGACATGGT

CATGCACCTGTACCA

GTA CGTGGACATGGT

CATCCACCTGTACCA

GTA GGTGGACATGGT

transition (T-A to C-G) transversion (T-A to G-C)

CATCACCTGTACCA

GTAGTGGACATGGT

deletionCATGTCACCTGTACCA

GTA C AGTGGACATGGT

insertion

base pair substitutions

transition: pyrimidine to pyrimidine

transversion: pyrimidine to purine

normal sequence

deletions and insertions can involve oneor more base pairs

Spontaneous mutations can be caused by tautomers


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p y

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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Cytosine

Cytosine

Guanine

Adenine

• cytosine mispairs with adenine resulting in a transition mutation

Normal tautomeric form

Rare imino tautomeric form

Mutation is perpetuated by replication


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utat o s pe petuated by ep cat o

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

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

AC• 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 Gand

C G

C

GC

Aand

C G

T A

Chemical mutagens


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g

Deamination by nitrous acid


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Derivation by hydroxylamine

The formation of a quarternary nitrogen destabilizes the

deoxyriboside bond and the base is released from deoxyribose

Alkylation by dimethyl sulfate causes depurination


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Attack by oxygen radicals


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Thymine dimer formation by UV light

Summary of DNA lesions


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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)

Mechanisms of Repair


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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 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


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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

Excision repair (base or nucleotide)


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ATGCUGCA TTGATAG

TACGGCGTAACTATC

thymine dimer

AT AG

TACGGCGTAACTATC

ATGCCGCATTGAT AG

TACGGCGTAACTATC

ATGCCGCATTGATAG

TACGGCGTAACTATC

excinuclease

DNA polymerase b

DNA ligase

(~30 nucleotides)

ATGCUGCATTGA

TACGGCGTAACT

ATGC GCATTGA

TACGGCGTAACT

AT GCATTGA

TACGGCGTAACT

deamination

ATGCCGCATTGA

TACGGCGTAACT

ATGCCGCATTGA

TACGGCGTAACT

uracil DNA glycosylase

repair nucleases

DNA polymerase b

DNA ligase

Base excision repair Nucleotide excision repair

Deamination of cytosine can be repaired


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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

Deamination of 5-methylcytosine cannot be repaired

Defects in DNA repair or replication


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• Xeroderma pigmentosum

• Ataxia telangiectasia

• Fanconi anemia

• Bloom syndrome• Cockayne syndrome

DNA repair activity

L i f e

s p a n

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 replicationAll are associated with a high frequency of chromosome


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g q y

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

dna mutation 3

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