Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity.
Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this polypeptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion.
Masutani
C
C
Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Japan.
Araki
M
M
Yamada
A
A
Kusumoto
R
R
Nogimori
T
T
Maekawa
T
T
Iwai
S
S
Hanaoka
F
F
D016590:Aphidicolin / Q000494:pharmacology; D001483:Base Sequence; D002457:Cell Extracts; D002478:Cells, Cultured; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency / Q000302:isolation & purification* / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D054306:Dideoxynucleotides; D005033:Ethylmaleimide / Q000494:pharmacology; D005347:Fibroblasts; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D010957:Plasmids / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000494:pharmacology; D011740:Pyrimidine Dimers / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics; D013942:Thymine Nucleotides / Q000494:pharmacology; D014983:Xeroderma Pigmentosum / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology
D002457:Cell Extracts; D003838:Deoxyadenine Nucleotides; D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D011740:Pyrimidine Dimers; D013942:Thymine Nucleotides; D015636:Magnesium Chloride; D016590:Aphidicolin; D004259:DNA-Directed DNA Polymerase; C017216:2',3'-dideoxythymidine triphosphate; C026600:2'-deoxyadenosine triphosphate; D005033:Ethylmaleimide