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, including double-strand DNA 3'-exonuclease, apurinic/apyrimidinic endonuclease, 3'-phosphatase, and 3'-phosphodiesterase. The carboxyl-terminal third of Rrp1, homologous to Escherichia coli exonuclease III, is sufficient to repair oxidative and alkylation-induced DNA damage in vivo. 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-BuO2H-induced DNA damage. Thr-462 and Lys-463 are highly conserved residues found in a cluster of 5 conserved amino acids (LQETK), while Leu-484 is poorly conserved. Gln-460 Glu-461, Thr-462, and Lys-463 and Leu-484 were altered by site-directed mutagenesis using 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. E461A has a 30-fold reduction in AP endonuclease and is MMS-sensitive, but all other mutants have near-normal AP endonuclease and are MMS-resistant. Glu-461 appears to be essential for the nuclease function for Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.
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Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Huang
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Sander
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D029721:Drosophila Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D017931:DNA Primers; D029721:Drosophila Proteins; D009713:Nucleotidyltransferases; C061900:Rrp1 protein, Drosophila