BINDING OF NUCLEAR PROTEINS ASSOCIATED WITH MAMMALIAN DNA-REPAIR TO THE MITOMYCIN-C DNA INTERSTRAND CROSS-LINK

Mitomycin C (MMC) is a DNA crosslinking agent that is used in cancer c hemotherapy. Unlike the DNA crosslinks formed by cisplatin or psoralen , which significantly distort the DNA helix, the MMC crosslink does no t significantly disturb the B-DNA helical structure. Nonetheless, MMC interstrand crosslinks and total MMC adducts are rapidly removed in vi vo. We investigated whether mammalian nuclear proteins can recognize a nd bind to a model 23 bp DNA duplex containing a single MMC lesion. El ectrophoretic mobility shift assays identified two complexes in nuclea r extracts from rodent cell lines and three complexes in human cell li nes, containing proteins that appeared to specifically recognize the M MC interstrand crosslink. Nuclear extracts from normal and excision re pair-defective mutant Chinese hamster ovary (CHO) cell lines, From hum an Xeroderma Pigmentosum (XP) complementation group A and E cell lines , and a Fanconi's Anemia cell line were also examined. The UV-20 CHO l ine, defective in ERCC-1, was missing one of the two rodent complexes. Two of the three human complexes were also absent in the XPA human ce ll line and the intensity of the third complex was significantly dimin ished. Based on these results, a model for MMC crosslink recognition i s proposed in which ERCC-1 and XPA each participate in formation of on e or more multimeric complexes on the crosslinked DNA and XPA also aid s in the Formation, but is nor a component of a higher molecularweight multimeric complex that may contain ERCC-1. (C) 1998 Wiley-Liss, Inc.