Role and regulation of homologous recombination in response to DNA double strand breaks and replication stress in Saccharomyces cerevisiae

Homologous recombination (HR) is a key pathway to maintain genomic integrity from one generation to another (meiosis) and during ontogenic development in a single organism (DNA repair). Recombination is required for the repair or tolerance of DNA damage and the recovery of stalled or broken replication forks. However, recombination is also potentially dangerous as it can lead to gross chromosomal rearrangements and potentially lethal intermediates. For this reason, recombinational events must be strictly regulated depending on the organism, cell type, cell-cycle stage, chromosomal region, as well as the type and level of genotoxic stress. HR participates in repair of DNA double-strand breaks (DSBs), which are among the most dangerous kinds of DNA lesions. Alternatively to HR, DSBs can be repaired also by non-homologous end joining (NHEJ). While NHEJ directly relegates the broken DNA ends, HR uses homologous DNA sequences as a template to form recombinants that are either crossover or noncrossover with regard to flanking parental sequences. Furthermore, a DSB flanked by direct DNA repeats can be repaired by a particular HR pathway called single-strand annealing (SSA), which results in DSB repair with concomitant deletion of one repeat and of the intervening sequence. All HR processes initiate with extensive 5’ to 3’ end-processing (a process referred to as 5’-3’ resection) of the broken ends to yield 3’-ended single-stranded DNA (ssDNA) tails, which are bound by Replication Protein A (RPA). RPA is then displaced by Rad51 to form nucleoprotein filaments that can catalyse homologous pairing and strand invasion. The choice between NHEJ and HR pathways is tightly regulated during the cell cycle. Eukaryotic cell division cycle comprises a series of events, whose ordering and correct progression depends on the oscillating activity of cyclin-dependent kinases (Cdks), which safeguard timely duplication and segregation of the genome. It has been demonstrated that Cdks promotes DNA repair by homologous recombination (HR) restricting this process to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. It has been demonstrated that Cdks promotes DNA repair by homologous recombination (HR). Cdk (Cdk1 in Saccharomyces cerevisiae) activity initiates HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps was unknown. To address this question, we explored the Cdk1 requirement in the execution of different HR processes in S. cerevisiae. In order to bypass the Cdk1 requirement for resection we analyzed cells lacking Yku heterodimer and/or the checkpoint protein Rad9, which are known as negative regulators of DSB resection. We showed that yku70Δ cells, which accumulate ssDNA at the DSB ends independently of Cdk1 activity, are able to repair a DSB by SSA in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by SSA and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes. As crossovers during mitotic cell growth have the potential for deleterious genome rearrangements when the sister chromatid is not used as repair template, this additional function of Cdk1 in promoting crossovers can provide another safety mechanism to ensure genome stability. Cells are particularly vulnerable to DNA damage during DNA replication because virtually all forms of DNA damage block DNA replication causing replication stress. Homologous recombination has pivotal roles in maintenance of genome integrity also during replication stress. HR machinery must be coordinated with S-phase checkpoint and replisome progression in order to ensure its stability during DNA synthesis. Among factors involved in both HR and replisome stability there is the conserved eterotrimeric complex MRX (Mre11–Rad50–Xrs2) in yeast, MRN (Mre11–Rad50–Nbs1) in mammals. This complex has different roles in DNA metabolism, as it is able to bind DNA and exerts architectural and catalytic functions. It has been demonstrated that this complex is important during DNA synthesis since it is essential in vertebrate and its disruption causes sensitivity to the replication inhibitor hydroxyurea (HU) in yeast. To better understand why MRX deficient cells are sensitive to replication stress we performed a genetic screening searching for spontaneous extragenic mutations that suppress the HU sensitivity of mre11Δ cells. We discovered that recessive mutation in TRR1 gene was able to partially suppress the HU sensitivity of mre11 strain. TRR1 encode for cytoplasmic thioredoxin reductase (Trr1), a key regulatory enzyme which cooperate with thioredoxins (Trx1 and Trx2) to forming a system involved in regulation of ribonucleotide reductase enzyme and in cellular response against oxidative. We found two mutated alleles of TRR1 (trr1-2 and trr1-6) each of one with a single base pair substitution which caused the amino acid substitutions A18D and I116S in trr1-2 and trr1-6 respectively. Both this mutation are in the Trr1 FAD binding domain and involve residues that are highly conserved in thioredoxin reductases from different organisms. We demonstrated that these mutated alleles encode for loss of function variants of thioredoxin reductase enzyme. Since thioredoxin system has a fundamental role in regulation of ribonucleotide reductase enzyme we tried to modulate the amounts of deoxyribonucleotide (dNTP) in order to understand if HU sensitivity suppression of mre11Δ cells could be linked to dNTPs levels during cell cycle progression. We showed that nor the increase neither the reduction of dNTPs amounts were responsible for the mre11Δ HU sensitivity suppression. As most of Trr1 functions required thioredoxins activities we tested if S. cerevisiae thioredoxins (Trx1 and Trx2) were involved in the mre11Δ cells HU sensitivity suppression. We demonstrated that loss of function mutations in TRR1 gene allele caused an increase in the amounts of both Trx1 and Trx2. However, we showed that variation in thioredoxins levels were not responsible for suppression of mre11Δ HU sensitivity. Then, we found that trr1 mediated HU sensitivity suppression was not specific for mre11Δ cells but was able to suppress the HU sensitivity of several mutants defective in HR pathway (rad51Δ, rad52Δ and sae2Δ) but not that of checkpoint mutants (mec1Δ and mrc1Δ). This demonstrated that Trr1 activity is deleterious during replication stress in the absence of functional recombination machinery. Although HU sensitivity of checkpoint mutants was not suppressed by loss of Trr1 function we showed that S-phase checkpoint was not involved in trr1 mediated HU sensitivity suppression of recombination mutants. In order to test if Trr1 activities were dangerous in cells defective in HR machinery which undergoes to replication stress we demonstrated that loss of functions of Trr1 partially prevented both the formation and accumulation of DSB lesions in HR mutants during HU treatment. Finally, we showed that loss of functions of Trr1 increased the frequency of nuclear division events of recombination mutants released from HU treatment. Thus, we concluded that thioredoxin reductase activities are dangerous during replication stress in the absence of recombination. We proposed that thioredoxin reductase inactivation could promote some HR alternative mechanisms or prevents the accumulation of HR substrates improving the nuclear division of recombination mutants which undergoes to replication stress.