DNA Repair

DNA Damage Tolerance and Repair

1-Dealing with Problems occurring during DNA replication

• Mutations resulting from errors made during DNA replication Mismatch Repair Pathway 

• Ribonucleotides incorporated during DNA replication  

2- Dealing with Problems coming after DNA replication resulting from DNA damage

• Non exhaustive list of Damages

• Not treated:-Double-stranded break repair-Transcription coupled DNA repair

• Strategies and mechanisms of DNA damage tolerance and repair:Bypass/Translesion DNA polymerasesDirect ReversalBase Excision RepairNucleotide Excision Repair

10-2

10-6

10-8-10-9

Editing

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Increasing Replication Fidelity by mismatch repair

template

New strand

How to differentiate between right and wrong Parental and new strand ?

!?

NH2

N3

N1

N7

N9

dR

O

OH OH

S+CH3

COO-+3HN

:

A

S-adenosyl methionine

New strand !

CH3CH3

DNA methylation is delayed after replication

2)

DNA methylation1)

GATCCTAG

Dam methylase

CH3

3HC

CTAGGATC

CH3

CH3

ATP

ADP+Pi

ATP

ADP+Pi

MutL/MutS MutH

Cleavage by MutH

CH3

DNA sliding throughMutL/MutS

CH3

CH3 CH3

Ligation

DNA unwinding byHelicase II (MutU/UvrD)

Exonuclease VII (5’-->3’) orExonuclease I (3’-->5’)

CH3

Methyl-directed mismatch repair in prokaryotes

Uvr genes = genes that promote UV Resistance

Mut genes = When these genes are mutated, bacteriashow increased rates of mutations

Filling the gap

The Problem of ribonucleotide incorporation by DNA polymerases

• DNA polymerases discriminate deoxy vs riboNTP, but not at 100%

• [rNTPs] >> [ dNTPs] in vivo -> this leads to riboNTP incorporation in newly synthesized DNA

Enzymes that deal with removal of RNA in Okazaki fragments also removeriboNTPs mis-incorporated into DNA :Rnase H, Fen1, DNA Pol.I

dd d d

aa

a a

e

ee

e

(PNAS 107, p4950, 2010)

Post-replicative DNA Damages that need to be repaired or dealt with

- Hydrolysis of glycosidic bond(Depurination)

- Alkylation of basesMethylation of guanine N6

G-> O6meG

-Pyrimidine dimers (UV light)

- Deamination of bases•Spontaneous •Chemically induced

•C->U•5 meC -> T•A->HX

- Oxidative damages•G -> 8 oxoguanine•Strand Break

- Bulky DNA adducts

Induction of Pyrimidine Dimers by UV light

PDB ID = 1SM5

Spontaneous Deaminations

A --> H10-9/24 hours

G --> X10-9/24 hours

C --> U10-7/24 hours:100 events/dayfor a mammaliancell

If Uracil were a naturalbase, the DNA repair machinerywould not know whether theseUracil are “normal” uracil that come from incorporation by polymerase, or “non-natural”uracil coming from deaminationof cytosines.Since there is no way to discri-minate between these, the genetic systems did not selecturacil as a natural base (exceptions)

Why Uracil was not selected as a natural base in DNA:

A C A T G G

T G U A U C

The problem of Uracil in DNA

Due to incorporation of dUTP by polymerase

Due to spontaneous

Deamination of C->U

5’ 3’

3’ 5’

Chemical Sources and Genetic Consequences of Deaminations

=

SpontaneousDepurinations

1/105 in 24 hours:10,000 events/day

for a mammalian cell

Chemical Sources and mechanism of alkylations

Consequences of O6-meG For Replication :

O2

O2-

1e-

2H+H2O2

1e-

-OH + .OH

2H2OCytC oxidase

Oxidative damage of DNA

- Source of oxidative agents

2O2-+2H+ H2O2 +O2

2H2O2

superoxide

dismutase

catalase2H2O+ O2

.OH

No cellularNeutralization

-> Main source ofOxidative agent

- Neutralization of reactive species

N

N

N NH2

O

NH H2O2

-OH

Guanine

5-formylUracil

Thymine

Consequences for Nucleotides:

Consequences for Nucleic Acids: Strand Breaks

(bad for DNA replication)

Deoxyribose Ribose

N

N

N NH2

O

NHO

H

8-oxo Guanine• Fenton Chemistry (Metals)

• Respiratory Chain:

G-C

DNA replication

H2O2

-OH

8-oxo-guanine generates replication block or G-C -> T:A transversions after DNA Replication

J Am Chem Soc. 2005 Oct 12;127(40):13906-18

8xoG•C

8xoG•A

T-A8xoG•A

DNA Polymerases tend to incorporate A opposite to 8-oxoG because of the tendency of 8-oxoG to switch in the syn conformation; A is the only nt that can form a base pair with syn8-oxoG whose geometry resembles that of a Watson-Crick base pair

Bulky DNA adducts caused by:

cigarette smoke

diesel engine exhaust

cooking/broiling of food

block DNA

Replication

DNA repairand tolerance

Strategies& Enzymes3- Base excision repair

(deamination, alkylation, oxidation of bases) • Uracil-N glycosylase • 8-oxoG glycosylase

2- Direct Reversal of Damage(alkylation of bases, pyrimidine dimers) • Photolyase reversion of Y dimers • Dealkylation of guanines by suicidal MGMTase • Dealkylation of 1mA and 3mC by AlkB (not shown)

4- Nucleotide excision repair(pyrimidine dimers, bulky DNA adducts) • Bacteria: UvrA, UvrB, UvrC, Helicase II (UvrD)• DNA pol. I, DNA ligase• Eukaryotes : Xeroderma pigmentosum proteins, TFIIH

1- Bypass of lesions: avoids DNA replication stalls • bypass of DNA damage by translesion DNA Polymerases -> not a “repair” but is used to prevent DNA replication blocks

Primer-

-X*

75nt-X*X X X*

Primer (41nt) 5’-AGGTemplate (75nt) 3’-TCCGTAXAATG--5’

Pol dPol

Bypass of 8-oxoG lesionsby a specialized eukaryoticDNA polymerase (Pol

X = Guanosine

X * = 8-Oxo Guanosine

<- Bypass product

Block of polymerization at 8-oxoG

Primer Extension Assayto map template replication by the twoDNA polymerases

Bacterial and Eukaryoticcells possess multiple translesion polymerasesthat are capable of bypassing DNA lesions

http://www.pathology.unc.edu/faculty_labs/vaziri_lab/image012.jpg

(4) Pol.h binds to mono-ubiquitinated PCNA and performs replicative bypass of damaged DNA, preserving replication fork movement.

1) A DNA lesion (red) causes stalling of the replicative DNA Pol.d.

3) Rad18 monoubiquitinates PCNA at stalled replication forks.

Switch between Replicative and Translesion DNA polymerases involves PCNAUbiquitination and prevents stalling of replication at the sites of DNA damage

2) The E3 ubiquitin ligase Rad18 guides Pol.h (aTLS DNA polymerase) to stalled replication forks.

PCNA

PCNA

PCNA

Photoreactivation

Repair ofpyrimidine dimers

h damage

DNA photolyase

Photoreactivating enzyme with 2 chromophores

e.g. 5-deazaflavin(or N5N10methyleneTHF) FADH-

h300-500nm

Direct Reversal:

• The “inactivated” enzyme serves as a transcription factor to induceexpression of DNA repair genes -> amplifies the cellular response to DNA damage

• Mutations of Human Homologues of O6MGMT linked to cancer : maintaining DNA information is required for tumor suppression

Dealkylation of guanines by Methyl Guanine Methyl Transferase (MGMT)

Base Excision Repair:General strategy

damaged base

+

DNA glycosylase/glycosidaseapurinic or apyrimidinic site

AP endonuclease Cuts strand at AP site(APurinic or Apyrimidic)leaves 5’ terminal deoxyribose P moiety

Second cleavage (1nt further) by another enzyme

DNA Pol + DNA ligase

OHP

OHP

Probably removes several nts by nick translation

Cleaves glycosidic bondat damaged base

1) Two sequential enzymatic activities are involved on two different enzymes:

2) The same polypeptide carries both glycosylase & AP endonuclease activities Ex: 8-OxoG DNA glycosylase (hOGG1)

Ex: Uracil N-glycosylase cleaves glycosidicbonds of deoxyuridine but does not have APendonuclease activity – needs another enzyme

Two types of base excision repair mechanisms

A) Pure glycosylase/glycosidase

thenB) AP endonuclease

Typically one enzyme cleaves the glycosidic bond of the damaged base, thenthe phosphodiester backbone is cleaved by an endonuclease specific for siteslacking a base (AP sites)

Structure and activity of uracil N-glycosylase

PDB ID = 2OXM

DNA Repair Strategies for 8-oxoG damage

OGG1 = glycosidase for 8-oxoG

MUTYH = glycosidase forA’s misincorporated in front ofthe 8-oxoG’s

David et al., Nature 447, 941 - 950 (2007)-Figure 1

The oxoG base is stacked between Phe 319 and Cys 253. Residues Gly 42, Gln 315 and two water molecules hydrogen bond to the Watson–Crick and Hoogsteen faces of the lesion base.

The cytosine paired opposite oxoG is recognized by H-bonding Interactions with Arg 154 and Arg 204, and an additional H bond with Asn 149.

How does hOGG1 recognize 8-OxoG damages ?

Bruner et al., Nature 403, 859 - 866 (24 February 2000)-Figure 6

recognition of 8-OxoG recognition of unpaired C

PDB ID = 1EBM

Nucleotide Excision Repair in Prokaryotes

Uvr = UV ResistanceWhen mutated, genes coding for Uvr proteins show an decrease in UV resistance

Nucleotide Excision Repairin Eukaryotes(Y dimer, bulky adducts)

XP = Xeroderma pigmentosum

TFIIH = basal RNA Polymerase II transcription factor H; contains XPB & XPD subunits

important becauseit links DNA repair totranscription