Human nucleotide excision nuclease removes thymine dimers from DNA by incising the 22nd phosphodiester bond 5' and the 6th phosphodiester bond 3' to the photodimer.
By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate consisting of a plasmid containing four thymidine dimers at unique locations, and deoxyribonucleoside 5'-[alpha-thio]triphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by I2, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer.
Huang
J C
JC
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Svoboda
D L
DL
Reardon
J T
JT
Sancar
A
A
D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D004258:DNA Polymerase III; D004260:DNA Repair; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011740:Pyrimidine Dimers; D004247:DNA; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; C036385:endodeoxyribonuclease uvrABC