title: Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position.
abstract: Many carcinogenic as well as chemotherapeutic agents cause covalent linkages between complementary strands of DNA. If unrepaired, DNA crosslinks are blocks to DNA replication and transcription and therefore represent potentially lethal lesions to the cell. Genetic studies of Escherichia coli have demonstrated that the repair enzyme ABC excision nuclease, coded for by the three unlinked genes, uvrA, uvrB, and uvrC, plays a crucial role in DNA crosslink repair. To study the molecular events of ABC excision nuclease-mediated crosslink repair, we have engineered a DNA fragment with a psoralen-DNA interstrand crosslink at a defined position, digested this substrate with pure enzyme, and analyzed the reaction products on DNA sequencing gels. We find that the excision nuclease cuts only one of the two strands involved in the crosslink, incises the crosslink by hydrolyzing the ninth phosphodiester bond 5' and the third phosphodiester bond 3' to the furan-side thymine of the crosslink, and does not produce double-strand breaks at any significant level. Based on these data, we present a model by which ABC excision nuclease initiates crosslink repair in vivo.
mesh_terms: D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005363:Ficusin / Q000378:metabolism*; D011564:Furocoumarins / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation
chemical_list: D029968:Escherichia coli Proteins; D011564:Furocoumarins; D004247:DNA; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D005363:Ficusin