A molecular genetic dissection of the evolutionarily conserved N terminus of yeast Rad52

Genetics. 2002 Jun;161(2):549-62.

Abstract

Rad52 is a DNA-binding protein that stimulates the annealing of complementary single-stranded DNA. Only the N terminus of Rad52 is evolutionarily conserved; it contains the core activity of the protein, including its DNA-binding activity. To identify amino acid residues that are important for Rad52 function(s), we systematically replaced 76 of 165 amino acid residues in the N terminus with alanine. These substitutions were examined for their effects on the repair of gamma-ray-induced DNA damage and on both interchromosomal and direct repeat heteroallelic recombination. This analysis identified five regions that are required for efficient gamma-ray damage repair or mitotic recombination. Two regions, I and II, also contain the classic mutations, rad52-2 and rad52-1, respectively. Interestingly, four of the five regions contain mutations that impair the ability to repair gamma-ray-induced DNA damage yet still allow mitotic recombinants to be produced at rates that are similar to or higher than those obtained with wild-type strains. In addition, a new class of separation-of-function mutation that is only partially deficient in the repair of gamma-ray damage, but exhibits decreased mitotic recombination similar to rad52 null strains, was identified. These results suggest that Rad52 protein acts differently on lesions that occur spontaneously during the cell cycle than on those induced by gamma-irradiation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / physiology
  • Amino Acid Sequence
  • Conserved Sequence
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / physiology
  • Evolution, Molecular
  • Gamma Rays
  • Molecular Sequence Data
  • Mutation
  • Rad52 DNA Repair and Recombination Protein
  • Recombination, Genetic
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / physiology
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / physiology
  • Sequence Alignment
  • Sequence Analysis, Protein

Substances

  • DNA-Binding Proteins
  • RAD52 protein, S cerevisiae
  • Rad52 DNA Repair and Recombination Protein
  • Saccharomyces cerevisiae Proteins
  • Alanine