Tight Regulation of Srs2 Helicase Activity Is Crucial for Proper Functioning of DNA Repair Mechanisms

Abstract

Proper DNA damage repair is one of the most vital and fundamental functions of every cell. Several different repair mechanisms exist to deal with various types of DNA damage, in various stages of the cell cycle and under different conditions. Homologous recombination is one of the most important repair mechanisms in all organisms. Srs2, a regulator of homologous recombination, is a DNA helicase involved in DNA repair, cell cycle progression and genome integrity. Srs2 can remove Rad51 from ssDNA, and is thought to inhibit unscheduled recombination. However, Srs2 has to be precisely regulated, as failure to do so is toxic and can lead to cell death. We noticed that a very slight elevation of the levels of Srs2 (by addition of a single extra copy of the SRS2 gene) leads to hyper-sensitivity of yeast cells to methyl methanesulfonate (MMS, a DNA damaging agent). This effect is seen in haploid, but not in diploid, cells. We analyzed the mechanism that controls haploid/diploid sensitivity and arrived to the conclusion that the sensitivity requires the activity of RAD59 and RDH54, whose expression in diploid cells is repressed. We carried out a mutational analysis of Srs2 to determine the regions of the protein required for the sensitization to genotoxins. Interestingly, Srs2 needs the HR machinery and its helicase activity for its toxicity, but does not need to dismantle Rad51. Our work underscores the tight regulation that is required on the levels of Srs2 activity, and the fact that Srs2 helicase activity plays a more central role in DNA repair than the ability of Srs2 to dismantle Rad51 filaments.

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

“…Several studies have shown that Tid1 is differently expressed in haploid and diploid cells (Bronstein et al, 2018;de Godoy et al, 2008;Galitski et al, 1999). We confirmed this differential expression by comparing the Tid1 protein levels in haploid and diploid cells (Figure 3A, 3B).…”

The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

“…Δ srs2 mutants are more sensitive to DNA damage as diploid cells than as haploid cells. Consistent with a more central role of HR repair in diploids ( 34 , 45 ), Δsrs2 diploids are more sensitive to the DSB-forming agent zeocin. We show that, in contrast, a diploid strain homozygous for the srs2 ( 1-850 ) allele is as proficient as the wt parent for growth in the presence of DNA-damaging agents, with the possible exception of MMS, where a barely detected defect can be seen ( Fig.…”

“…Although many times described as an “antirecombinase,” Srs2 is essential for DSB repair by HR ( 27 ); thus, its activity is both pro- and antirecombinational ( 44 ). Moreover, even Srs2 alleles that lack the region required for interactions with Rad51 or PCNA (or with any of the proven Srs2 interactors) are still proficient in promoting synthesis-dependent strand annealing (SDSA) over crossover resolution ( 79 ) and perfectly complement the sensitivity of Δsrs2 mutants to DNA damage ( 34 , 37 , 38 , 80 this work). The new srs2 allele [ srs2 ( 1-850 )], which lacks the entire C terminus and has only the DNA helicase domain ( 15 ), is unable to interact with Rad51 ( 15 ) or with any of the known partners of Srs2 (PCNA, Rad51, Nej1, Mre11, Sgs1, Esc2, Ubc9, Siz1, Siz2, Mus81, Rad5, and Rad18 [ 23 , 40 – 44 ]).…”

“…Our recent study provided evidence that Srs2 has functions that are independent of its role in the eviction of Rad51 and of its interaction with PCNA. Neither the Rad51 interaction region, PIM (PCNA-interacting motif), nor SIM (SUMO-interacting motif), which allow recruitment of Srs2 to SUMOylated PCNA, are required to deal with DNA damage caused by methyl methanesulfonate (MMS) ( 34 ).…”

“…Ploidy seems to affect the fundamental regulation of the pathways involved in dealing with DNA replication stress. Haploids rely more on the DDT pathways, whereas diploids seem to rely more on HR ( 34 , 45 ). It seems that PCNA and its modifications affect the regulation of DNA repair during replication, depending on the ploidy of the cell.…”