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Endogenous DNA Damage. from Marnett and Plastaras, Trends Genet . 17 , 214 (2001). Biological Molecules are Labile. RNA is susceptible to hydrolysis. Reduction of ribose to deoxyribose gives DNA greater stability. N-glycosyl bond of DNA is more labile.

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slide1

Endogenous DNA Damage

from Marnett and Plastaras, Trends Genet. 17, 214 (2001)

slide2

Biological Molecules are Labile

RNA is susceptible to hydrolysis

Reduction of ribose to deoxyribose gives DNA greater stability

N-glycosyl bond of DNA is more labile

DNA damage occurs from normal cellular operations

and random interactions with the environment

slide3

Spontaneous Changes that Alter DNA Structure

deamination

oxidation

depurination

from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-46

slide4

Hydrolysis of the N-glycosyl Bond of DNA

from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-47

Spontaneous depurination results in loss of 10,000 bases/cell/day

Causes formation of an AP site – not mutagenic

slide5

Deamination of Cytosine to Uracil

from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-47

Cytosine is deaminated to uracil at a rate of 100-500/cell/day

Uracil is excised by uracil-DNA-glycosylase to form AP site

slide6

5-Methyl Cytosine Deamination is Highly Mutagenic

Deamination of 5-methyl

cytosine to T occurs rapidly

- base pairs with A

5-me-C is a target for

spontaneous mutations

from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-52

slide7

Deamination of A and G Occur Less Frequently

A is deaminated to HX – base pairs with C

G is deaminated to X – base pairs with C

from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-52

slide8

Oxidative Damage of DNA

Oxidative damage results from aerobic metabolism, environmental toxins,

activated macrophages, and signaling molecules (NO)

Compartmentation limits oxidative DNA damage

slide9

Oxidation of Guanine Forms 8-Oxoguanine

The most common mutagenic

base lesion is 8-oxoguanine

guanine

8-oxoguanine

from Banerjee et al., Nature 434, 612 (2005)

slide10

Repair of 8-oxoG

Replication of the 8-oxoG strand

preferentially mispairs with A

and mimics a normal base pair

and results in a G-to-T transversion

8-oxoguanine DNA glycosylase/

b-lyase (OGG1) removes 8-oxo-G

and creates an AP site

MUTYH removes the A opposite 8-oxoG

slide11

Oxidation of dNTPs are Mutagenic

cGTP is oxidized to 8-OH-dGTP and is misincorporated opposite A

MutT converts 8-OH-dGTP to 8-OH-dGMP

slide12

UV-Irradiation Causes Formation of Thymine Dimers

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-38

slide13

Nonenzymatic Methylation of DNA

Formation of 600 3-me-A residues/cell/day are caused by S-andnosylmethionine

3-me-A is cytotoxic and is repaired by 3-me-A-DNA glycosylase

7-me-G is the main aberrant base present in DNA and

is repaired by nonenzymatic cleavage of the glycosyl bond

slide14

Effect of Chemical Mutagens

Nitrous acid causes deamination of C to U and A to HX

U base pairs with A

HX base pairs with C

slide15

Spores Use Strategies to Overcome Intrinsic Instability of DNA

DNA exists in an A-like conformation and is bound

to proteins that reduce the rate of depurination

Lack of dNTPs in spores prevents DNA repair before germination

Extensive DNA repair occurs upon spore germination

slide16

Repair Pathways for Altered DNA Bases

from Lindahl and Wood, Science 286, 1897 (1999)

slide17

Direct Repair of DNA

Photoreactivation of pyrimidine dimers by photolyase restores the original DNA structure

O6-methylguanine is repaired by removal of methyl group by MGMT

1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation

slide18

Base Excision Repair of a G-T Mismatch

BER works primarily on modifications

caused by endogenous agents

At least 8 DNA glcosylases are

present in mammalian cells

DNA glycosylases remove

mismatched or abnormal bases

AP endonuclease cleaves 5’ to AP site

AP lyase cleaves 3’ to AP site

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-36

slide19

Mechanism of hOGG1 Action

from David, Nature434, 569 (2005)

hOGG1 binds nonspecifically to DNA

Contacts with C results in the extrusion of corresponding base in the opposite strand

G is extruded into the G-specific pocket,

but is denied access to the oxoG pocket

oxoG moves out of the G-specific pocket, enters the

oxoG-specific pocket, and excised from the DNA

slide20

Nucleotide Excision Repair in Human Cells

NER works mainly on helix-distorting

damage caused by environmental mutagens

The only pathway to repair thymine dimers

in humans is nucleotide excision repair

Mutations in at least seven XP genes

inactivate nucleotide excision repair

and cause xeroderma pigmentosum

XPC recognizes damaged DNA

Helicase activities of XPB and XPD of

TFIIH create sites for XPF and XPG cleavage

An oligonucleotide containing the

lesion is released and the gap is filled

by POL d or e and sealed by LIG1

from Lindahl and Wood, Science286, 1897 (1999)

slide21

Transcription-coupled Repair

Repair of the transcribed strand of active genes is

corrected 5-10-fold as fast as the nontranscribed strand

All the factors required for NER are required for transcription-coupled repair except XPC

The arrest of POL II progression at a lesion served as a damage recognition signal

Recruitment of NER factors also involves CS-A and CS-B

slide22

Nucleotide Excision Repair Pathway in Mammals

Cockayne’s Syndrome and Trichothiodystrophy

are multisystem disorders defective in

transcription-coupled DNA repair

slide23

Mismatch Repair in E. coli

Newly replicated DNA is hemimethylated

MutS binds to mismatch and recruits MutL

Activates endonuclease activity of MutH

and nicks the nearest unmethylated GATC

Recruits MutU (helicase) and exonucleases

DNA pol III fills in the gap

slide24

Mismatch Repair in Human Cells

MSH2 and MSH6 bind to mismatch-

containing DNA and distinguish between

the template and newly synthesized strand

MMR complex identifies newly synthesized

strand by the presence of a 3’-terminus

MutLa introduces random nicks

at distal sites on the same strand

EXO1 at 5’-side of the mismatch activates

a 5’-3’ exonuclease and removes mismatch

The gap is filled in by DNA polymerase

and DNA ligase

Defective mismatch repair is the primary

cause of certain types of human cancers

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-37

slide25

Causes of and Responses to ds Breaks

DSBs result from exogenous insults

or normal cellular processes

DSBs result in cell cycle

arrest, cell death, or repair

Repair of DSBs is by

homologous recombination

or nonhomologous end joining

from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

slide26

ATM Mediates the Cell’s Response to DSBs

DSBs activate ATM

ATM phosphorylation of p53,

NBS1 and H2AX influence cell

cycle progression and DNA repatr

from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

slide27

Repair of ds Breaks by Homologous Recombination

ssDNAs with 3’ends are formed and

coated with Rad51, the RecA homolog

Rad51-coated ssDNA invades the

homologous dsDNA in the sister chromatid

The 3’-end is elongated by DNA polymerase,

and base pairs with ss 3-end of the other broken DNA

DNA polymerase and DNA ligase fills in gaps

from Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31

slide28

Repair of ds Breaks by Nonhomologous End Joining

KU heterodimer recognizes

DSBs and recruits DNA-PK

Mre11 complex tethers ends

together and processes DNA ends

DNA ligase IV and

XRCC4 ligates DNA ends

from van Gent et al., Nature Rev.Genet. 2, 196 (2001)

slide29

Structure of Rad50 and the Mre11 Complex

Mre11 complex maintains

genome integrity and

participates in HR and NHEJ

AT-like syndrome and Nijmegen

breakage syndrome are caused by

mutation in MRE11 or NBS1

Dimerization domain of Rad50 keeps

DNA fragments within close proximity

from de Jager and Kanaar, Genes Dev.16, 2173 (2002)

slide30

XRCC4

X-ray-repair-cross-complementing defective repair in Chinese hamster mutant 4

slide31

The SCID Mouse is Defective in DSB Repair

The SCID mouse carries a mutation preventing

the production of mature B and T cells

The phenotype is a defect in the development of the

immune system and hypersensitivity to ionizing radiation

The phenotype is caused by a mutation in DNA-PK

slide32

Translesion Replication by DNA Polymerase V

Translesion DNA synthesis occurs

in the absence of Pol III

Translesion DNA polymerases are

error prone and exhibit weak processivity

Most of the mutations caused by DNA

damaging agents are caused by TLR

TLR protects the genome from gross rearrangements

Pol V is regulated by LexA and the SOS response

from Livneh, J.Biol.Chem. 276, 25639 (2001)

slide33

Expandable Repeats Form Unusual DNA Structures

One of the complementary strands is

more structure-prone than the other

Unusual structural features

predispose them to instability

Stalling of lagging strand

synthesis disrupts coordination

with leading strand synthesis

from Mirkin, Nature447, 932 (2007)