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REPAIR OF DNA

Types of repair Direct repair, base excision repair, and nucleotide excision repair Introduction to DSB repair

Chromosomes of organisms undergo many types of DAMAGE

Some is beneficial – evolution, immune system

Accumulation of certain lesions can have severe effects onDNA metabolism

Cells contain a number of mechanisms that can alter or cor-rect damage

DNA Repair

Interest in the field is intense because of the implica-tion of DNA damage in the etiology of cancer, aging, and mental retardation

Ding et al. (2018) Cell 173, 305-320.

64% of cancer predisposition genes affecting genome integrity are DNA repair genes-green

bars on Circos plot

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DNA Repair genes and cancer

DNA is not a stable chemical

Subject to modification:

Errors in DNA replication, recombination, and repair

Chemically reactive nucleotides at ambient temperaturesand pH

Interaction with chemical and physical agentsSpontaneous, endogenousExogenous – environmental

Can lead to breaks in the DNA

Copyright ©2003 by the National Academy of Sciences

Vilenchik, Michael M. and Knudson, Alfred G. (2003) Proc. Natl. Acad. Sci. USA 100, 12871-12876

Fig. 1. Flow chart of the rates of production of EDSBs from SS lesions in mammalian cells under normal conditions

1 EDSB/108 bp

Daniel, R. et al. J. Biol. Chem. 2004;279:45810-45814

Retroviral infection induces formation of {gamma}H2AX foci

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Lesions that are removed by repair, replaced by recom-bination, or lead to mutations and cell death

1)  Missing base -acid and heat depurination -removal by enzymes-glycosylases

2)  Altered base -alkylating agents-electrophilic rea-gents seek out nucleophilic substituents, N3 of A, N7 of G,other ring nitrogens and exocyclic Os3)  Incorrect base -misinsertion

-defective proofreading-defective MMR-deamination of C to U; CG to TA

4) Deletion or insertion - frameshift, primer dislocation5) Cyclobutyl dimer - can’t form hydrogen bonded pairs6) Crosslinks - interstrand and intrastrand and protein/DNA

cisplatin, psoralen, mitomycin C, aldehyde

100% ds DNA

98% dsDNA 2% ssDNA

Depurination 18,000 18,000

Depyrimidination

600 600

C deaminat. 100 500

5-MeC deaminat.

10 50

Endogenous Lesions Arising and repaired per 24 h in a mammalian cell:

Friedberg et al. (2006) DNA Repair and Mutagenesis, ASM Press

Sunlight

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Repair Pathways

1.  Isolate mutants that can’t repair-make a model

2.  Find enzymes affected by the mutations

3.  Determine their substrates and activities

4.  See whether they match your modelDirect Reversal of Damage

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Excision Repair

Excision repair relies on redundancy of informationin the DNA

Damage is removed and then the gap is repairedusing the complementary strand as template

Two enzymatic pathways: Base Excision Repair Nucleotide Excision Repair

Specific recognition of the primary chemical or photochemical lesion initiates repair and is rate limiting

Short patch, removes nucleotides, single bases, not oligonucleotides

Long patch, removes a few nucleotides

BER

BER

Limited substrate range

Uracil removal: Deamination of cytosine to uracil or misincorporation of dUTP

Short patch: -DNA glycosylases-AP-endonucleases/lyases-polymerase-ligase

Long patch:DNA glycosylases-AP-endonucleases-polymerase-FEN1/PCNA-Ligase-same mechanism as OFP

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

oOr lyase

Pol beta

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Uracil N-glycosylase Uracil is the main source of abasic sites in DNADue primarily to incorportation of dUMP into DNA rather than to deamination of Uracil Uracil glycoslyase- Small, no cofactorRecognize damage in a sea of DNACrystal structure of UDG shows the base is pulled out of the helix and sits in a pocket on the enzyme where specific amino acid side chains hydrolyze the C-1’ (glycosyl) bond Based on co-crystals with amino-uracil or thymine trinucleotide

• AP endonuclease usually cleaves 5’ to missing base and usually leaves a 3’ OH and 5’ phosphate

• AP cleavage also occurs by AP-lyase Cleaves 3’ to the missing base β elimination lyases yield 3’ unsaturated aldehyde

and 5’ phosphate δ elimination lyases yield 5’ phosphate, 3’ phosphate

• Lyases are part of some polymerases, such as pol β and of some glycosylases (which cleave the sugar/base glycosidic linkage to remove the base)

INCISION ACTIVITIES

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Cleavage of the AP site to give a gap

Friedberg et al. DNA Repair and Mutagenesis,ASM Press 2006

3’-OH 5’-P Missing base

• AP endonuclease usually cleaves 5’ to missing base and usually leaves a 3’ OH and 5’ phosphate

• AP cleavage also occurs by AP-lyase- β-elimination 3’ to the missing base β elimination lyases- yield 3’ unsaturated aldehyde

and 5’ phosphate δ elimination lyases-5’ phosphate, 3’ phosphate

• Lyases are part of some polymerases, like pol β or perhaps of some glycosylases (which cleave the sugar/base glycosidic linkage to remove the base)

INCISION ACTIVITIES

N-glycosylase Uracil- Small, no cofactorRecognize damage in a sea of DNACrystal structure of UDG shows the base is pulled out of the helix and sits in a pocket on the enzyme where specific amino acid side chains hydrolyze the C-1’ (glycosyl) bond Based on co-crystals with amino-uracil or thymine trinucleotide

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Incorporation of dUMP during replication and its removal by uracil DNA glycosylases is a critical source of

endogenous AP sites in DNA. apn1 apn2 rad1 triple mutant is inviable because of its incapacity to repair AP sites and related 3'-blocked single-strand breaks deletion of the UNG1 gene encoding the uracil DNA glycosylase suppresses the lethality of the apn1 apn2 rad1 mutant. MAG1, OGG1, or NTG1 and NTG2 encoding DNA glycosylases involved in the repair of alkylation or oxidation damage does not suppress lethality Uracil is the main source of abasic sites in DNA

Uracil can arise in DNA by cytosine deamination or by the incorporation of dUMP during replication Overexpression of the DUT1 (dUTPASE) gene suppresses the lethality of the apn1 apn2 rad1 mutant Uracil in DNA comes mainly from incorporation of dUTP during DNA replication- overexpression of dUTPase suppresses lethality of apn1apn2rad1.

How does Uracil arise in DNA?

BER and disease

In human cells, DNA polymerase β repairs gap/breastcancer mutants

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Class 2: Short patch mismatch repair - mutY- glycosylases thatrecognize oxidative damage that causes 8-oxoguanine, SAM methlylation, and other alkylations such as N3 of A and N7 of G

Alterations in the major groove and charged molecules

8-oxoG, pairs with A, gives a G to T -transversion

ROS (reactive oxygen species) Hydroxyl radical,OH· (formed by H2O2 and Fe++), H2O2; Superoxide (O2

-), More intimate molding of the enzyme on the DNA

Class 3: Large enzymes with both glycosylase and endonuclease- iron-sulfur enzymes

NER Nucleotide Excision Repair Pathways

1.  Isolate mutants that can’t repair-make a model

2.  Find enzymes affected by the mutations

3.  Determine their substrates and activities

4.  See whether they match your model

5.  E. coli-uvrA, uvrB, uvrC, uvrD, ssb, pol A, lig

6.  CHO mutants-complementation by human genes, ERCC,excision repair cross complementation

Xeroderma pigmentosum, XPA-XPG, XPV(variant)

7. Yeast - rad mutants

uvrA,B,C,D, ssb, polA, lig E. coli

RAD 1, 2, 3, 4, 10, 14, 25 Yeast

XPG F, G, D, C, ERCC1, A, B Human

More complicated recognition and repairMore extensive substrate range

UV survival curves

NER

Dose (J/m2)

Survival (%) WT

Mutant

Unscheduled DNA synthesis (UDS), incorporation of 3H-Tdrafter UV irradiation-autoradiographyImmunofluorescence-antibody to specific lesion

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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Dual Incision Model Model: Dual incision model

5’ 3’

T-TRAD1,10(XPF)

RAD2(XPG)

5’ 3’

T-T

Bacterial in vitro system

UvrA-ATP-dependent damage recognition UvrA a matchmaker: makes UvrB recognize and bind damaged DNA better-UvrB a helicase

UvrA2UvrB unwinds, bends, and causesa conformational change - UvrA leaves (catalytic)

UvrC recruited and UvrC is activated to a 3’ nuclease and then to a 5’ nuclease, after rearrangement (ATP required but not hydrolysis)

UvrD, a helicase, releases the oligomer during DNA synthesis by Pol1

Ligase seals

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Enzyme system hydrolyzes two phosphodiester bonds,One on either side of the lesion

Helicase generates an oligonucleotide carrying the damage

After release from the duplex, the gap is filled inand ligated to complete the reaction

Repair patches are 12-13 NT in bacteria or 27 to 29 in mammals, therefore the cuts are precise

Substrates: thymine dimers (UV), benzo(a) pyrene-guanineadducts (smoking), thymine-psoralen adducts,guanine-cisplatin adducts (chemotherapy), mismatches

Repair everything, sometimes detrimental, need regulation

Lesion is recognized by XPC-RAD23B complex, an ATP depen-dent binding factor -(hR23B identified by ability to complementan XPC defect in a cell-free DNA repair system containingUV damaged SV40 minichromosomes)

Creates open complex-local melting at site of lesion, recruits other factors (like the role of dnaA, ORC (?))

XPB and XPD recruited. Helicases that unwind around the lesion, more DNA unwound 5’ to lesion

ssDNA is stabilized by XPA and RPA (3 subunit SSB)

XPA interacts with XPB,XPD, and XPF-ERCC1 (no mutantsin ERCC1, probably essential)

General genomic repair (GGR)

A single trimer of RPA binds to the 30 bp of opened DNA, in a specific orientation, to bind XPG (a FEN1 family nuclease) on 3’ side and ERCC1-XPF on the5’ side of the lesion

XPG 3’ and XPF 5’ recognize ssDNA/dsDNA junction

RPA confers strand specificity to damaged strand cutting

Gap filling by PCNA, RFC, RPA, and pol ε or pol δ

Sealing by ligase I

Χοµπλετελψ ρεχονστιτυτεδ ιν ϖιτρο ωιτη εξτραχτσ ανδ πυριφιεδ χοµπονεντσ: extract or purified proteins, UV irradiated DNA substrate, assay for incorporation of radiolabeled dNTPs, analyze product by electrophoresis and autoradiography

Egly (2004)JBC279,19074

Distortion recognition

XPA-damage location and recruit

XPC/hR23B

RPA

XPB and XPD

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XPB-XPD helicases

XPF and XPG nucleases

XPB and XPD helicases (Rad25 and Rad3 in yeast)

Subunits of txn factor TFIIH, which contains at least seven otherpolypeptides, TFIIH is recruited to damage with XPA

XPC and XPG may be part of TFIIH (Rad10 and Rad2), part of3’ structure specific nuclease

TCR (transcription coupled repair)

RNA polymerase recognizes damage and recruits repairTFIIH recognizes damage and recruits the nucleases

Keeps transcription from being interrupted by damage in the template

Up to 8 h to transcribe the globin gene

Preferential repair of the transcribed strand

Van Brabant et al.(2000) Annu. Rev.Hum. Genet. 1:409-59

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Deficiencies in repair enzymes cause disease

1968- Cleaver-Nature- Xeroderma pigmentosum, a RARE skin cancer,found mostly in people with chronic exposure to the sun, had defects in NER-XPA-G,XPV,Fibroblasts from patients are UV sensitive

Cockayne’s Syndrome- growth and mental retardation,photosensitivity, transcription coupling defective-not an overall repair defect, repair of expressed genes CSA, CSB, XPB, XPD

TTD-Trichothiodystrophy-brittle hair, mental retardation, XPB,XPD,XPG

These mutations don’t implicate NER in cancer predispositionCairns proposed therefore that mutations don’t cause cancer, but he didn’t recognize the other pathways BER, mismatch, DSB-also concluded endogenous damage more important in cancer

Most chemotherapy relies on drugs that cause lesions repaired by these pathways

Repair status of tumor cell may affect therapyDifference in repair between normal and cancer cell couldpotentially make drugs more (or less) effective on tumorthan on normal cell. Eg. HMG proteins bind lesions and inhibit repair and are differentially expressed;PARPi’s

Study of coupling of repair to replication, transcription and theCell cycle-Checkpoints- biochemical pathways that serveas surveillance mechanisms to detect damage and slow thecell cycle until damage is dealt with-ATM, ATR, RAS and p53 in humansSOS in bacteria

General genomic repair (GGR)