Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage

L. Gu, S. M. Huang, M. Sander

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 of Rrp1. Lys-463 and, to a lesser extent, Thr-462 influence the substrate specificity of the Rrp1 nuclease.

Original languageEnglish (US)
Pages (from-to)32685-32692
Number of pages8
JournalJournal of Biological Chemistry
Volume269
Issue number51
StatePublished - Dec 1 1994

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DNA-(Apurinic or Apyrimidinic Site) Lyase
Methyl Methanesulfonate
DNA Damage
Drosophila
spleen exonuclease
Repair
Phosphoric Diester Hydrolases
Amino Acids
Escherichia coli
DNA
Exodeoxyribonucleases
Mutagenesis
Endonucleases
Alkylation
Mutant Proteins
Substrate Specificity
Site-Directed Mutagenesis
Phosphoric Monoester Hydrolases
Plasmids
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Single amino acid changes alter the repair specificity of Drosophila Rrp1. Isolation of mutants deficient in repair of oxidative DNA damage. / Gu, L.; Huang, S. M.; Sander, M.

In: Journal of Biological Chemistry, Vol. 269, No. 51, 01.12.1994, p. 32685-32692.

Research output: Contribution to journalArticle

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