Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
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.
Kazantsev
A
A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7260, USA.
Mu
D
D
Nichols
A F
AF
Zhao
X
X
Linn
S
S
Sancar
A
A
D001483:Base Sequence; D002474:Cell-Free System; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics; D051716:Replication Protein A; D014983:Xeroderma Pigmentosum / Q000235:genetics*
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A