Single and Doublestrand

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Single and Double-strand Break Repair
Andrew Pierce Microbiology, Immunology and
Molecular Genetics Toxicology University of
Kentucky
TOX 780
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Single-strand Nicks
Any covalent disruption in the phosphodiester
backbone in ONE of the two DNA strands in
double-helical DNA
Reactive oxygen species Base hydrolysis Base
excision repair 1.5 x 105 per cell per day
Ligatable needs a 5'-PO4 end and a 3'-OH
end Otherwise, need some end processing AP-endonuc
lease, polynucleotide kinase, DNA-polymerase b
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Base Excision Repair
APE1 (AP endonuclease-1) Can cleave to the 5'
side of abasic sites and damaged termini to leave
a 3' OH
Polb Can cleave to the 3' side of abasic
sites Has dRP-lyase activity
Marti TM, Fleck O. DNA repair nucleases Cell Mol
Life Sci. 2004 Feb61(3)336-54
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XRCC1 Interactions
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XRCC1 Holds It Together
DNA pol b
XRCC1 (N-term)
Nicked DNA
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Single-strand Nick Repair Summary
The single-strand nick lesion is recognized by
PARP which recruits XRCC1 or by XRCC1
directly XRCC1 forms a protein scaffold and
recruits APE (AP-endonuclease) and PNK
(polynucleotide kinase) to trim the DNA
ends XRCC1 recruits DNA pol b for additional
trimming and nucleotide replacement XRCC1
recruits DNA ligase III to restore covalent
continuity to the repaired DNA strand Mouse
knockouts of any of XRCC1, APE, DNA pol b are
embryonic lethal XRCC1-deficient cell lines show
10x elevation in levels of sister chromatid
exchange conversion of single-strand nicks to
double-stranded breaks
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Double-strand Breaks Challenge Genome Stability
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Mammalian End-joining Proteins
Ku 70
Ku 80
DNA PKCS
Ku 80
Ku 70
DNA PKCS
Ku 80
Ku 80
Ku 70
Ku 70
DNA PKCS
XRCC4
Ku 70
Ku 80
Lig IV
Lig IV
Ku 70
Ku 80
DNA PKCS
XRCC4
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Ku70/80 Heterodimer Binds DNA Double-strand
Ends In Preparation for End-joining
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Other Proteins That May Play an End-Processing
Role
Artemis opens hairpins XPG (Fen1) (XRCC5) flap
endonuclease, 5' terminated single-stranded
flaps ERCC1/XPF (ERCC4) flap endonuclease, 3'
terminated single-stranded flaps
Fen-1 (P. furiosus)