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PMID: 9351836 Published · ppublish English Journal Article

Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.

The EMBO journal ·Vol. 16 ·No. 21 ·1997-11-03 ·Pages 6559-73

Evans E, Moggs JG, Hwang JR, Egly JM, Wood RD

Abstract

During nucleotide excision repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core nucleotide excision repair factors. Opening is followed by dual incision, with the 3' cleavage made first.

MeSH Terms
Adenosine Triphosphate/metabolism Cells, Cultured Cisplatin/pharmacology DNA/drug effects,metabolism DNA Damage DNA Helicases/metabolism DNA Ligases/metabolism DNA Repair/physiology DNA, Single-Stranded/metabolism DNA-Binding Proteins/metabolism Endonucleases HeLa Cells Humans Macromolecular Substances Models, Genetic Nuclear Proteins Point Mutation Proteins/metabolism Replication Protein A Substrate Specificity Transcription Factor TFIIH Transcription Factors/chemistry,metabolism Transcription Factors, TFII Transcription, Genetic Xeroderma Pigmentosum/enzymology,genetics,pathology Xeroderma Pigmentosum Group A Protein Xeroderma Pigmentosum Group D Protein
Chemicals
DNA excision repair protein ERCC-5 DNA, Single-Stranded DNA-Binding Proteins Macromolecular Substances Nuclear Proteins Proteins RPA1 protein, human Replication Protein A Transcription Factors Transcription Factors, TFII XPA protein, human Xeroderma Pigmentosum Group A Protein xeroderma pigmentosum group F protein XPBC-ERCC-3 protein Transcription Factor TFIIH XPC protein, human Adenosine Triphosphate DNA ERCC1 protein, human Endonucleases DNA Helicases Xeroderma Pigmentosum Group D Protein ERCC2 protein, human DNA Ligases Cisplatin
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Evans E
Imperial Cancer Research Fund, Clare Hall Laboratories, Hertfordshire, UK.
Moggs J G
Hwang J R
Egly J M
Wood R D
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
DOI
10.1093/emboj/16.21.6559
Published
1997-11-03
Pages
6559-73
Language
English
Region
England
NLM ID
8208664
PMCID
PMC1170260
Subset
IM
External Links
PubMed source DOI: 10.1093/emboj/16.21.6559 PMC Full Text View journal details
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