Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange

Larry H. Thompson, Kerry W. Brookman, Nigel J. Jones, Susan A. Allen, Anthony V. Carrano

Research output: Contribution to journalArticle

359 Citations (Scopus)

Abstract

We describe the cloning and function of the human XRCC1 gene, which is the first mammalian gene isolated that affects cellular sensitivity to ionizing radiation. The CHO mutant EM9 has 10-fold-higher sensitivity to ethyl methanesulfonate, 1.8-fold-higher sensitivity to ionizing radiation, a reduced capacity to rejoin single-strand DNA breaks, and a 10-fold-elevated level of sister chromatid exchange compared with the CHO parental cells. The complementing human gene was cloned from a cosmid library of a tertiary transformant. Two cosmid clones produced transformants that showed ≈100% correction of the repair defect in EM9 cells, as determined by the kinetics of strand break repair, cell survival, and the level of sister chromatid exchange. A nearly full-length clone obtained from the pcD2 human cDNA expression library gave ≈80% correction of EM9, as determined by the level of sister chromatid exchange. Based on an analysis of the nucleotide sequence of the cDNA insert compared with that of the 5′ end of the gene from a cosmid clone, the cDNA clone appeared to be missing ≈100 bp of transcribed sequence, including 26 nucleotides of coding sequence. The cDNA probe detected a single transcript of ≈2.2 kb in HeLa polyadenylated RNA by Northern (RNA) blot hybridization. From the open reading frame and the positions of likely start sites for transcription and translation, the size of the putative XRCC1 protein is 633 amino acids (69.5 kDa). The size of the XRCC1 gene is 33 kb, as determined by localizing the endpoints on a restriction endonuclease site map of one cosmid clone. The deduced amino acid sequence did not show significant homology with any protein in the protein sequence data bases examined.

Original languageEnglish (US)
Pages (from-to)6160-6171
Number of pages12
JournalMolecular and Cellular Biology
Volume10
Issue number12
StatePublished - 1990
Externally publishedYes

Fingerprint

Sister Chromatid Exchange
DNA Breaks
Molecular Cloning
Cosmids
Clone Cells
Complementary DNA
Genes
Ionizing Radiation
Ethyl Methanesulfonate
Single-Stranded DNA Breaks
Proteins
CHO Cells
Transcription Initiation Site
DNA Restriction Enzymes
Gene Library
Northern Blotting
Open Reading Frames
Organism Cloning
Amino Acid Sequence
Cell Survival

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Thompson, L. H., Brookman, K. W., Jones, N. J., Allen, S. A., & Carrano, A. V. (1990). Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. Molecular and Cellular Biology, 10(12), 6160-6171.

Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. / Thompson, Larry H.; Brookman, Kerry W.; Jones, Nigel J.; Allen, Susan A.; Carrano, Anthony V.

In: Molecular and Cellular Biology, Vol. 10, No. 12, 1990, p. 6160-6171.

Research output: Contribution to journalArticle

Thompson, LH, Brookman, KW, Jones, NJ, Allen, SA & Carrano, AV 1990, 'Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange', Molecular and Cellular Biology, vol. 10, no. 12, pp. 6160-6171.
Thompson, Larry H. ; Brookman, Kerry W. ; Jones, Nigel J. ; Allen, Susan A. ; Carrano, Anthony V. / Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. In: Molecular and Cellular Biology. 1990 ; Vol. 10, No. 12. pp. 6160-6171.
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