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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)

53

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