INITIATION OF DNA INTERSTRAND CROSS-LINK REPAIR IN HUMANS - THE NUCLEOTIDE EXCISION-REPAIR SYSTEM MAKES DUAL INCISIONS-5' TO THE CROSS-LINKED BASE AND REMOVES A 22-NUCLEOTIDE-LONG TO 28-NUCLEOTIDE-LONG DAMAGE-FREE STRAND

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with norm al ones, using the complementary strand as a template. Interstrand cro ss-links pose a unique challenge to the DNA repair machinery because b oth strands are damaged. To study the repair of interstrand cross-link s by mammalian cells, we tested the activities of cell extracts of wil d-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link We found that in contrast to monoadducts, which are removed by dual inci sions bracketing the lesion, the cross-link causes dual incisions, bot h 5' to the cross-link in one of the two strands. The net result is th e generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link This gap may act as a recombinogenic signal to initiate cro ss-link removal.