Evidence for a novel DNA damage binding protein in human cells.
We describe a novel DNA damage binding activity in nuclear extracts from a normal human fibroblast cell strain. This protein was identified using electrophoretic mobility shift assays of immunopurified UV-irradiated oligonucleotide substrates containing a single, site-specific cyclobutane pyrimidine dimer or a pyrimidine (6-4) pyrimidinone photoproduct. Compared with the (6-4) photoproduct, which displayed similar levels of binding in double and single-stranded substrates, the protein showed somewhat lower affinity for the cyclobutane dimer in a single-stranded oligonucleotide and negligible binding in double-stranded DNA. The specificity and magnitude of binding was similar in cells with normal excision repair (GM637) and repair-deficient cells from xeroderma pigmentosum groups A (XP12RO) and E (XP2RO). An apparent molecular mass of 66 kDa consisting of two subunits of approximately 22 and approximately 44 kDa was determined by Southwestern analysis. Cell cycle studies using centrifugal cell elutriation indicated that the binding activity was significantly greater in G1 phase compared with S phase in a human lymphoblast cell line. Gel supershift analysis using an anti-replication protein A antibody showed that the binding protein was not antigenically related to the human single-stranded binding protein. Taken together, these data suggest that this activity represents a novel DNA damage binding protein that, in addition to a putative role in excision repair, may also function in cell cycle or gene regulation.
Ghosh
R
R
Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, Smithville 78957, USA.
Peng
C H
CH
Mitchell
D L
DL
D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000528:radiation effects; D004247:DNA / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts; D016193:G1 Phase; D006801:Humans; D008214:Lymphocytes; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D016196:S Phase; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D004247:DNA