Incision of a 1,3-intrastrand d(GpTpG)-cisplatin adduct by nucleotide excision repair proteins from yeast.
The protein machinery responsible for nucleotide excision repair (NER) is highly conserved from yeast to man. NER can be reconstituted with purified proteins, and the incision sites around a defined DNA lesion have been defined to the nucleotide level in a mammalian NER system. Here, we reconstitute NER in yeast whole cell extracts, as well as with partially purified yeast NER components. We show that NER activity can be isolated partly as a large protein complex, and map the sites of nucleotide incision around a cisplatin-induced DNA lesion. Our data indicate that yeast NER proteins excise an oligonucleotide of 23-26 bases containing the DNA lesion (rather than 26-30 bases as in humans), and that the 3' incision occurs around position 17 (rather than at position 9 as in humans).
Kong
Stephanie E
SE
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, UK.
Svejstrup
Jesper Q
JQ
D002457:Cell Extracts; D002945:Cisplatin / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004270:DNA, Circular / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism; D004720:Endonucleases / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases
D002457:Cell Extracts; D018736:DNA Adducts; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; C057270:cisplatin-DNA adduct; C073998:RAD2 protein, S cerevisiae; D012319:RNA Polymerase II; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes; D002945:Cisplatin