Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.
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.
Selby
C P
CP
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Sancar
A
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D001426:Bacterial Proteins / Q000302:isolation & purification; D002474:Cell-Free System; D002945:Cisplatin / Q000633:toxicity; D004249:DNA Damage; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics*; D005363:Ficusin / Q000633:toxicity; D066298:In Vitro Techniques; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D005363:Ficusin; D002945:Cisplatin