Involvement of the Saccharomyces cerevisiae HDF1 Gene in DNA Double-strand Break Repair and Recombination

Guenter J Mages,Heidi M Feldmann,Ernst-Ludwig Winnacker

doi:10.1074/jbc.271.14.7910

Abstract

The HDF1 protein of Saccharomyces cerevisiae shares biochemical properties and structural homology with the 70-kDa subunit of the human autoantigen Ku. The Ku protein, a heterodimer composed of a 70-kDa subunit and an 80-kDa subunit, has been identified as the regulatory subunit of the DNA-dependent protein kinase. This enzyme has recently been shown to be involved in DNA repair and recombination processes in mammalian cells. Here we show that hdf1-disrupted S. cerevisiae strains are strongly sensitive toward the radiomimetic antibiotic bleomycin. In addition, mating-type switching and rates of spontaneous mitotic recombination are strongly reduced. This phenotype is similar to that of mammalian cells lacking components of the DNA-dependent protein kinase holoenzyme, suggesting that HDF1 participates in and exerts equivalent functions in S. cerevisiae.

Highlights

DNA double-strand breaks (DSBs)1 are intermediates of recombination events in both pro- and eukaryotic cells
Mitotic Recombination Is Reduced by hdf1 Deficiency—Because the hdf1 mutation affects the rate of mating-type switching, which is a site-specific recombination event, we studied the effect of the hdf1 mutation on spontaneous mitotic recombination
This paper describes experiments that try to understand and explain the phenotype of the hdf1 mutation by studying its effects on bleomycin sensitivity, mating-type switching, and mitotic recombination frequencies

Summary

MATERIALS AND METHODS

Isogenic W303rad52-4D was generated by crossing W303␣ and XS5601C-1D1. ␣ segregants were tested for ura allele and twice back-crossed with W303-1A, resulting in W303rad52-4D. For the characterization of hdf rad double mutants, W303rad52-4D was crossed with W303␣U and spore clones of two complete tetratype tetrades (W303LU-5 to W303LU-8 and W303LU-17 to W303LU-20) were used for the assays. Complementation analysis of hdf mutants have been performed with the single copy vector pRS316 carrying the genomic XhoI–EcoRI fragment of the HDF1 gene. Mitotic recombination experiments were performed with strains or their derivatives generously provided by.

Relevant genotype

RESULTS

Findings

DISCUSSION