SINGLE AMINO-ACID CHANGES ALTER THE REPAIR SPECIFICITY OF DROSOPHILA RRP1 - ISOLATION OF MUTANTS DEFICIENT IN REPAIR OF OXIDATIVE DNA-DAMAGE

Drosophila Rrp1 has several tightly associated enzymatic activities, i ncluding double-strand DNA 3'-exo-nuclease, apurinic/apyrimidinic endo nuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-termi nal third of Rrp1, homologous to Escherichia coil exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vi vo. Using a screen for partial complementation of repair-deficient E., coli, we isolated three mutants of the nuclease domain of Rrp1: T462A , K463Q, and L484P, that protect against methyl methanesulfonate (MMS) -induced but not t-BuO(2)H-induced DNA damage, Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino aci ds (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-46 2, and Lys-463 and Leu-484 were altered by site-directed mutagenesis u sing a plasmid including the entire Rrp1 gene and mutant proteins were purified, Mutants of the three residues Glu-461, Thr-462, and Lys-463 demonstrate 8-200-fold lower phosphodiesterase specific activity than wild-type Rrp1., E46lA has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonucle ase and are MMS-resistant, Glu-461 appears to be essential for the nuc lease function of Rrp1. Lys-463 and, to a lesser extent, Thr-462 influ ence the substrate specificity of the Rrp1 nuclease.