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Virtual Free Radical School. DNA Oxidation: Basics of Repair. Karl E. Herbert, Ph.D. & Pratibha Mistry, Ph.D. Faculty of Medicine & Biological Sciences, University of Leicester, Leicester, UK. feedback: keh3@le.ac.uk. Overview. DNA damage base excision repair ( BER )

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dna oxidation basics of repair

Virtual Free Radical School

DNA Oxidation: Basics of Repair

Karl E. Herbert, Ph.D. &

Pratibha Mistry, Ph.D.

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

feedback: keh3@le.ac.uk

SFRBM Education Program

overview
Overview
  • DNA damage
  • base excision repair (BER)
  • global genome (GG) & transcription coupled (TC) nucleotide excision repair (NER)
  • sanitization of the deoxynucleotide pool

SFRBM Education Program

consequences of dna damage
Consequences of DNA damage

DNA DAMAGE

DNA REPAIR MECHANISMS

SHORT-TERM CONSEQUENCES

PHYSIOLOGICAL DYSFUNCTION

CELL DEATH

ABNORMAL GROWTH & METABOLISM

Decreased cellular proliferation

Genomic instability

Defective signalling pathways

Impaired protein/ gene expression

LONG-TERM CONSEQUENCES

Ageing

Cancer

Disease

SFRBM Education Program

types of oxidative dna damage
Types of oxidative DNA damage
  • purine & pyrimidine oxidation products
  • abasic (AP) sites
  • strand breaks
  • see Schafer, SFRBM Education Program presentation:
  • http://www.medicine.uiowa.edu/frrb/ VirtualSchool/Virtual.html

SFRBM Education Program

slide5
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

SFRBM Education Program

ber enzymes e g functional diversity i
BER enzymes: e.g. functional diversity I

MutM/fpg: formamidopyrimidine glycosylase

endo VIII/Nei: endonuclease VIII

OGG: 8-oxoguanine DNA glycosylase

NEIL: Nei endonuclease VIII-like

MPG: N-methylpurine-DNA glycosylase

AlkA: 3-methyladenine DNA glycosylase II

SFRBM Education Program

ber enzymes e g functional diversity ii
BER enzymes: e.g. functional diversity II

endo III : endonucleases III

Ntg: endonuclease III-like glycosylase

NTHL1: Nth endonuclease III-like 1

TDG: thymine-DNA glycosylase

SFRBM Education Program

short patch ber

ROS

OH

C

8-oxoG

T

A

C

Bi-functional glycosylase

8-oxoG

3’ -lyase

OH

C

T

A

C

A

A

A

A

A

G

G

G

G

G

T

T

T

T

T

C

C

C

C

C

G

G

G

G

G

APE

HO

HO

HO

HO

HO

OH

dGTP

C

C

A

T

DNA Pol

XRCC1

HO

OH

C

T

A

C

DNA ligase III

OH

C

G

T

A

C

Short-patch BER

SFRBM Education Program

ber of 8 oxog

NucleicAcids

8-oxoguanine

H

Reactive Oxygen Species Damage

hAPE:

-elimination

H

hOGG1:

-lyase

hOGG1:

glycosylase

BER of 8-oxoG

hAPE/HAP1/REF1: apurinic/apyrimidinic endonuclease

SFRBM Education Program

other ber proteins
Other BER Proteins

SFRBM Education Program

long patch ber

Single-strand break

HO

OH

C

T

C

T

A

G

A

G

C

A

G

T

C

G

PARP

XRCC1

HO

OH

C

T

C

T

A

G

A

G

C

A

G

T

C

G

PCNA

DNA Pol/

A

A

A

G

G

T

T

C

C

G

G

A

A

G

T

C

G

G

G

T

T

C

C

G

G

HO

HO

HO

HO

HO

OH

dNTP’s

C

C

G

C

G

G

A

C

T

C

C

C

A

C

A

C

A

G

C

T

T

T

A

G

T

C

G

FEN-1

OH

T

C

A

G

A

G

T

C

G

C

T

C

T

A

G

A

G

C

C

DNA ligase I

OH

T

C

A

G

A

G

T

C

G

Long-patch BER

SFRBM Education Program

dna repair pathways the prevention of mutations
DNA repair pathways & the prevention of mutations

Complexities of DNA repair pathways & replication…

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

Sanitization of the deoxynucleotide pool...

..specific enzymes prevent the incorporation of damaged deoxynucleotides opposite a template strand during DNA replication

SFRBM Education Program

complexities of repair pathways replication

8-oxoG:

8-oxoG:A

8-oxoG:A

T:A

hMYH

mismatch

A

e.g. NEIL1

8-oxoG

8-oxoG:C

e.g. NEIL1

8-oxoG

G:C

Complexities of repair pathways & replication..

“template strand”

misincorporation

oxidation

A/T/G

G:

C

good 

bad X

SFRBM Education Program

sanitization of the deoxynucleotide pool
Sanitization of the deoxynucleotide pool..

…prevents incorporation of 8-oxoG into DNA

MutT: nucleoside triphosphate pyrophosphohydrolase

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

hMTH1: human homolog of MutT

SFRBM Education Program

gg tc ner
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

SFRBM Education Program

gg tc ner16

Bulky lesion

Transcription-coupled

Global Genomic

5’

3’

3’

5’

5’

5’

3’

3’

XPC:hHR23B

CSA

RNA Pol II

‘DNA unwinding’: XPB, XPD

3’

3’

5’

5’

CSB

24-29mer repair product

XPF

XPG

TFIIH

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

5’

3’

XPA

3’

5’

RPA

3’

5’

5’

3’

5’

3’

3’

5’

DNA ligase

GG & TC NER

SFRBM Education Program

bibliography
Bibliography

Human 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: 1-8

Friedberg 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:

101-113

Hazra 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 SciUSA99: 3523-3528

Hoeijmakers JHJ (2001). Genome maintenance mechanisms for preventing cancer. Nature.411: 366-374

Krokan HE, Nilsen H, Skorpen F, Otterlei M and Slupphaug G (2000). Base excision repair of DNA in mammalian cells. FEBS Lett.476: 73-77

Lindahl T and Wood RD (1999). Quality control by DNA repair. Science.286: 1897-1905

Mitra 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 295-311

Sancar A (1996). DNA excision repair. Ann Rev Biochem.65: 43-81

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