MECHANISM OF TRACKING AND CLEAVAGE OF ADDUCT-DAMAGED DNA SUBSTRATES BY THE MAMMALIAN 5'-EXONUCLEASE TO 3'-EXONUCLEASE ENDONUCLEASE RAD2 HOMOLOG-1 OR FLAP ENDONUCLEASE-1

The mammalian 5'- to 3'-exonuclease/endonuclease, called RAD2 homologu e 1 or flap endonuclease 1, has a unique cleavage activity, dependent on specific substrate structure. On a primer-template, in which the pr imer has an unannealed 5'-tail, endonucleolytic cleavage near the anne aling point releases the tail intact. Entering at the 5'-end, the nucl ease tracks along the entire tail to the point of cleavage, Genetic an alyses suggest that this nuclease removes DNA adducts in vivo (Sommers , C. H., Miller, E. J., Dujon, B., Prakash, S., and Prakash, L. (1995) J. Biol. Chem. 270, 4193-4196). Micrococcal nuclease footprinting sho ws that after tracking the nuclease protects a region of the tail 25 n ucleotides long, adjacent to the cleavage site. Substrates with adduct s at specific locations were used to assess the mechanism of RAD2 homo logue 1 nuclease tracking and its ability to cleave modified DNA. Eith er a conventional cis-diamminedichloroplatinum (II) (CDDP) or a bulky CDDP derivative was placed within or beyond the region protected by th e nuclease. The nuclease cleaved the tail of both substrates. In contr ast, a CDDP adduct just adjacent to the expected cleavage point was in hibitory. A CDDP adduct at the very 5'-end of the tail was also cleave d. The nuclease could remove tails containing adducts on the sugar-pho sphate backbone. Apparently, the nuclease is designed to slide over va rious types of damage on single stranded DNA and then cut past the dam aged site.