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. 1991 Sep 15;88(18):8232–8236. doi: 10.1073/pnas.88.18.8232

Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.

C P Selby 1, A Sancar 1
PMCID: PMC52481  PMID: 1896474

Abstract

In eukaryotic and prokaryotic cells, activity transcribed genes and, in some instances, the template strand of these genes have been found to be repaired 2-10 times more rapidly than nontranscribed genes or the coding strand of transcribed genes. We demonstrate here gene- and template strand-specific repair synthesis in vitro by using an Escherichia coli cell-free extract and a plasmid carrying a gene with the strong tac promoter. Strand-specific repair of UV, 4'-hydroxymethyl1-4,5',8-trimethylpsoralen, and cis-dicholorodiammine platinum(II) damage was dependent upon transcription and a functional nucleotide excision repair system and was stimulated by 6% (wt/vol) polyethylene glycol. A defined system consisting of the transcription and repair proteins in highly purified form did not perform strand-specific repair; however, active fractions of extract conferred strand specificity to the defined system. Transcription-repair coupling activity was partially purified from extract by successive DEAE-agarose and gel filtration chromatography. The coupling factor is heat-labile, with an estimated Mr of 100,000.

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Selected References

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