SFRBM Education Program Herbert/Mistry 1DNA Oxidation: Repair 5/2003 Karl E. Herbert, Ph.D. &...

Herbert/Mistry 1 SFRBM Education Program

DNA Oxidation: Repair5/2003

Karl E. Herbert, Ph.D. & Pratibha Mistry, Ph.D.

Faculty of Medicine & Biological Sciences, University of Leicester, Leicester, UK

feedback: [email protected]: [email protected]

DNA Oxidation: Basics of Repair

Virtual Free Radical SchoolVirtual Free Radical School

SFRBM Education Program

Herbert/Mistry 2DNA Oxidation: Repair5/2003

Overview

• DNA damage

• base excision repair (BER)

• global genome (GG) & transcription coupled (TC) nucleotide excision repair (NER)

• sanitization of the deoxynucleotide pool



Consequences of DNA damage

LONG-TERM CONSEQUENCESLONG-TERM CONSEQUENCES

Ageing Cancer Disease

DNA REPAIR MECHANISMSDNA REPAIR MECHANISMS

SHORT-TERM CONSEQUENCESSHORT-TERM CONSEQUENCES

ABNORMAL GROWTH & METABOLISM

PHYSIOLOGICAL DYSFUNCTION

CELL DEATH

Decreased cellular

proliferation

Defective signalling pathways

Impaired protein/ gene expression

Genomic instability

DNA DAMAGE



Types of oxidative DNA damage

• see Schafer, SFRBM Education Program presentation:

http://www.medicine.uiowa.edu/frrb/ VirtualSchool/Virtual.html

• purine & pyrimidine oxidation products

• abasic (AP) sites

• strand breaks



BER

• non-bulky lesions, abasic sites & strand breaks are repaired by BER

• mono- or bifunctional lesion-specific glycosylases may initiate repair by the cleavage of the N-C1’ glycosidic bond between the base & deoxyribose-phosphate backbone leaving an abasic site

• following the removal of abasic sites, short or long-patch BER resumes resulting in the repair of a single or 2-8 nucleotide gap, respectively



BER enzymes: e.g. functional diversity I

Oxidized & ring opened purines:e.g. 8-oxoG, fapyG

E.coli S. cerevisiae H. sapiens

MutM (fpg)

endo VIII (Nei)

AlkA (not oxoG)

yOGG1&2

_ _ _ _

_ _ _ _

hOGG1

hNEIL1

hMPG

MutM/fpg: formamidopyrimidine glycosylaseendo VIII/Nei: endonuclease VIII OGG: 8-oxoguanine DNA glycosylaseNEIL: Nei endonuclease VIII-likeMPG: N-methylpurine-DNA glycosylase AlkA: 3-methyladenine DNA glycosylase II



BER enzymes: e.g. functional diversity II

Oxidized & ring opened pyrimidines: e.g. T-glycol, fapy

E.coli S. cerevisiae H. sapiens

endo III (Nth)

endo VIII (Nei)

AlkA _ _ _ _ _

Ntg1&2

_ _ _ _ _

_ _ _ _ _

_ _ _ _ _

hNTHL1

hNEIL1-3_ _ _ _

hTDG

endo III : endonucleases IIINtg: endonuclease III-like glycosylase NTHL1: Nth endonuclease III-like 1TDG: thymine-DNA glycosylase



Short-patch BERROS

C 8-oxoGA TC

OH

Bi-functional glycosylase

C A TC

OH

3’ -lyase8-oxoG

HO

C A TC

OH

G CT AG

HO

G CT AG

HO

G CT AG

HO

C A TC

OH

G CT AG

HO

APE

dGTP

DNA Pol

XRCC1

C GA TC

OH

DNA ligase III

G CT AG

HO



BER of 8-oxoG

O

OO N

N

N

N

O

N

O

HH

P

O O

O

O

O

N

N

N

O

P

O O

O

HH

PO

OO

8-oxoguanine

Rea

ctiv

e O

xyge

n S

pec

ies

Dam

age

hAPE:-elimination

hOGG1:-lyase

hOGG1:glycosylase

Nucleic Acids

H

H

hAPE/HAP1/REF1: apurinic/apyrimidinic endonuclease



Other BER Proteins

Removal of abasic sitesAPE1(HAP1, REF1)

AP endonuclease

Gap fillingPCNA Proliferating cell nuclear antigen

DNA pol DNA polymerase

Sealing the nickXRCC1 X-ray cross-complementing

protein & ligase accessory factor

LIG3 DNA ligase



Long-patch BERSingle-strand break

G CT AG

HO

C GA T

HO

C GA TC

OH

G CT AG

G CT AG

HO

C GA T

HO

C GA TC

OH

G CT AG

PARPXRCC1

dNTP’s

G CT AG

HO

C GA T

T

C

G

A

COH

G CT AG

DNA Pol/PCNA

C

G CT AG

HO

C GA T

OH

G CT AGT C GA CC

DNA ligase I

G CT AG

HO

C GA T

T

C

G

A

COH

G CT AGT C GA CC

T

C

G

A

C

FEN-1



DNA repair pathways & the prevention of mutations

..specific enzymes prevent incorrect base pairing ..specific enzymes prevent incorrect base pairing opposite DNA lesions during DNA replicationopposite DNA lesions during DNA replication

..specific enzymes prevent the incorporation of ..specific enzymes prevent the incorporation of damaged deoxynucleotides opposite a damaged deoxynucleotides opposite a

template strand during DNA replicationtemplate strand during DNA replication

Sanitization of the deoxynucleotide pool...

Complexities of DNA repair pathways & replication…



Complexities of repair pathways & replication..

8-oxoG:A

8-oxoG:C

hMYHmismatch

G:C

e.g. NEIL1

A

8-oxoG

8-oxoG:A

T:A

e.g. NEIL18-oxoG

good bad X

“template strand”misincorporation

8-oxoG: A/T/G

oxidation

CC G:



Sanitization of the deoxynucleotide pool..

MutT: nucleoside triphosphate pyrophosphohydrolase

NUDT1: nudix (nucleoside diphosphate linked moiety X)-type motif 1

hMTH1: human homolog of MutT

E.coli H. sapiens substrate

MutT NUDT1 (hMTH1) 8-oxodGTP

…prevents incorporation of 8-oxoG into DNA



GG & TC NER

•bulky & small lesions causing differing degrees of DNA distortion are removed by NER

•NER is implicated as a back-up system for BER

•different complexes initiate NER in transcribed & non-transcribed DNA

•NER involves the dual incision of unwound DNA either side of the lesion

by a multisubunit ATP-dependent nuclease & the release of a 24-29 oligomer

•the resultant gap is filled & sealed by the action of various components including DNA polymerases & ligases



GG & TC NER

3’

5’

5’

3’

‘Repair synthesis’: dNTP’s, PCNA, RFC, RPA, DNA Pol XRCC1

3’

5’

5’

3’

DNA ligase

24-29mer repair product

5’

3’

XPGXPF

XPA

TFIIH

RPA

3’

5’

Global Genomic Transcription-coupled

5’

3’

3’

5’

XPC:hHR23B

5’

3’

3’

5’

RNA Pol IICSA

CSB

5’

3’

3’

5’

Bulky lesion

‘DNA unwinding’: XPB, XPD



BibliographyHuman DNA Repair Enzymes:

http://www.cgal.icnet.uk/DNA_Repair_Genes.html Boiteux S and Radicella JP (2000). The human OGG1 gene: structure,

functions and its implication in the process of carcinogenesis. Arch Biochem Biophys. 377: 1-8Friedberg EC (1996). Relationships between DNA repair and transcription. Ann Rev Biochem. 65: 15-42 Hanawalt PC (1995). DNA repair comes of age. Mutat Res. 336:

101-113Hazra TK, Izumi T, Boldogh I, Imhoff B, Kow YW, Jaruga P, Dizdaroglu M and Mitra S (2002). Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA. Proc Natl Acad Sci USA 99: 3523-3528Hoeijmakers JHJ (2001). Genome maintenance mechanisms for preventing cancer. Nature. 411: 366-374Krokan HE, Nilsen H, Skorpen F, Otterlei M and Slupphaug G (2000). Base excision repair of DNA in mammalian cells. FEBS Lett. 476: 73-77Lindahl T and Wood RD (1999). Quality control by DNA repair. Science. 286: 1897-1905Mitra S (1999). Repair of oxidative DNA damage and aging; central role of AP-endonuclease. In: Dizdaroglu and Karakaya, eds. Advances in DNA Damage and Repair, Kluwer Academic, Plenum Publishers, New York. pp 295-311Sancar A (1996). DNA excision repair. Ann Rev Biochem. 65: 43-81

SFRBM Education Program Herbert/Mistry 1DNA Oxidation: Repair 5/2003 Karl E. Herbert, Ph.D. &...

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