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PMID: 8643521 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 93 ·No. 10 ·1996-05-14 ·Pages 5014-8

Kazantsev A, Mu D, Nichols AF, Zhao X, Linn S, Sancar A

Abstract

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

MeSH Terms
Base Sequence Cell-Free System DNA Damage DNA Repair/genetics DNA, Complementary/genetics DNA-Binding Proteins/genetics Escherichia coli/genetics Genetic Complementation Test HeLa Cells Humans In Vitro Techniques Molecular Sequence Data Mutation Recombinant Proteins/genetics Replication Protein A Xeroderma Pigmentosum/genetics
Chemicals
DNA, Complementary DNA-Binding Proteins RPA1 protein, human Recombinant Proteins Replication Protein A
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Kazantsev A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260, USA.
Mu D
Nichols A F
Zhao X
Linn S
Sancar A
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.93.10.5014
Published
1996-05-14
Pages
5014-8
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC39398
Subset
IM
Grants
NIGMS NIH HHS · GM32833 · United States
External Links
PubMed source DOI: 10.1073/pnas.93.10.5014 PMC Full Text View journal details
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