Role of Homologous Recombination Genes in Repair of Alkylation Base Damage by Candida albicans.

Toni Ciudad,Germán Larriba,Alberto Bellido,Encarnación Andaluz,Belén Hermosa

doi:10.3390/genes9090447

Abstract

Candida albicans mutants deficient in homologous recombination (HR) are extremely sensitive to the alkylating agent methyl-methane-sulfonate (MMS). Here, we have investigated the role of HR genes in the protection and repair of C. albicans chromosomes by taking advantage of the heat-labile property (55 °C) of MMS-induced base damage. Acute MMS treatments of cycling cells caused chromosome fragmentation in vitro (55 °C) due to the generation of heat-dependent breaks (HDBs), but not in vivo (30 °C). Following removal of MMS wild type, cells regained the chromosome ladder regardless of whether they were transferred to yeast extract/peptone/dextrose (YPD) or to phosphate buffer saline (PBS); however, repair of HDB/chromosome restitution was faster in YPD, suggesting that it was accelerated by metabolic energy and further fueled by the subsequent overgrowth of survivors. Compared to wild type CAI4, chromosome restitution in YPD was not altered in a Carad59 isogenic derivative, whereas it was significantly delayed in Carad51 and Carad52 counterparts. However, when post-MMS incubation took place in PBS, chromosome restitution in wild type and HR mutants occurred with similar kinetics, suggesting that the exquisite sensitivity of Carad51 and Carad52 mutants to MMS is due to defective fork restart. Overall, our results demonstrate that repair of HDBs by resting cells of C. albicans is rather independent of CaRad51, CaRad52, and CaRad59, suggesting that it occurs mainly by base excision repair (BER).

Highlights

Methyl-methane-sulfonate (MMS) is used for the analysis of pathways involved in repair/tolerance to methylation [1]
Methyl-methane-sulfonate generates methylated bases on dsDNA whose repair can cause nicks, gaps, and, indirectly, double-strand breaks (DSBs) that can engage in homologous recombination (HR) directly or cause fork stalling during the replication round [2–4]
We show that resting cells of C. albicans can repair heat dependent breaks (HDBs) in the absence of HR, whereas repair of HDBs by cycling cell was mostly dependent on efficient HR

Summary

Introduction

Methyl-methane-sulfonate (MMS) is used for the analysis of pathways involved in repair/tolerance to methylation [1]. Methyl-methane-sulfonate generates methylated bases on dsDNA whose repair can cause nicks, gaps, and, indirectly, double-strand breaks (DSBs) that can engage in homologous recombination (HR) directly or cause fork stalling during the replication round [2–4]. Methylated bases can be directly removed by DNA-methyl-transferases, the major repair pathway consists of a step-wise process known as base excision repair (BER) [1,5]. The apurinic/apyrimidic (AP) sites generated are removed by redundant AP endonucleases. Apn and Apn, which cleave 50 the AP-site to form nicks with a 50 desoxyribose phosphate (50 dRP). Removal of 50 dRP is carried out by the coordinated action of DNA polymerase (δ or ε) and the flap endonuclease Rad27/Fen, followed by ligation.

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Results

Conclusion