Evidence for a novel DNA damage binding protein in human cells

Rita Ghosh, Chuan Hsien Peng, David L. Mitchell

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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 ≃22 and ≃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.

Original languageEnglish (US)
Pages (from-to)6918-6923
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number14
DOIs
StatePublished - Jul 9 1996
Externally publishedYes

Fingerprint

DNA-Binding Proteins
DNA Damage
Oligonucleotides
DNA Repair
Carrier Proteins
Pyrimidinones
Replication Protein A
Cyclobutanes
Pyrimidine Dimers
cdc Genes
Xeroderma Pigmentosum
G1 Phase
Electrophoretic Mobility Shift Assay
S Phase
Cell Cycle
Proteins
Fibroblasts
Gels
Cell Line
Antibodies

Keywords

  • cell cycle
  • nucleotide excision repair
  • UV radiation

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Evidence for a novel DNA damage binding protein in human cells. / Ghosh, Rita; Peng, Chuan Hsien; Mitchell, David L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, No. 14, 09.07.1996, p. 6918-6923.

Research output: Contribution to journalArticle

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