Non-homologous end joining is important for repair of Cr(VI)-induced DNA damage in Saccharomyces cerevisiae

Hexavalent chromium is known to be a potent carcinogen that leads to many different DNA lesions, including DNA-protein crosslinks, and single- and double-strand breaks. In Saccharomyces cerevisiae, DNA double-strand breaks are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ) repair pathways. Here, we show that mutants deficient in NHEJ (yku70Delta, rad50Delta, dnl4Delta, mre11Delta, xrs2Delta) of S. cerevisiae are more sensitive to Cr(VI) toxic effects than wild-type cells. Also, a deletion mutant of SAE2 showed a similar sensitivity to Cr(VI), even though it has no apparent direct role in NHEJ. We also found that double mutants in HR and NHEJ (yku70Delta/rad52Delta, rad50Delta/rad52Delta, dnl4Delta/rad52Delta, mre11Delta/rad52Delta, xrs2Delta/rad52Delta) are synergistically more sensitive to Cr(VI) exposure than any of the single mutants, indicating that both repair pathways are involved in the repair of Cr(VI)-induced lesions. Finally, when the NHEJ mutants were exposed to Cr(VI) under anaerobic growth conditions, Cr(VI) toxicity was suppressed.     

Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage

Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies.     

Mdt1 facilitates efficient repair of blocked DNA double-strand breaks and recombinational maintenance of telomeres

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3'-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Delta also hypersensitizes partially recombination-defective cells to camptothecin-induced 3'-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Delta cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair "clean" endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Delta leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Delta causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance.     

Effects of mutations in DNA repair genes on formation of ribosomal DNA circles and life span in Saccharomyces cerevisiae

A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.     

Pathway utilization in response to a site-specific DNA double-strand break in fission yeast

We have examined the genetic requirements for efficient repair of a site-specific DNA double-strand break (DSB) in Schizosaccharomyces pombe. Tech nology was developed in which a unique DSB could be generated in a non-essential minichromosome, Ch(16), using the Saccharomyces cerevisiae HO-endonuclease and its target site, MATa. DSB repair in this context was predominantly through interchromosomal gene conversion. We found that the homologous recombination (HR) genes rhp51(+), rad22A(+), rad32(+) and the nucleotide excision repair gene rad16(+) were required for efficient interchromosomal gene conversion. Further, DSB-induced cell cycle delay and efficient HR required the DNA integrity checkpoint gene rad3(+). Rhp55 was required for interchromosomal gene conversion; however, an alternative DSB repair mechanism was used in an rhp55Delta background involving ku70(+) and rhp51(+). Surprisingly, DSB-induced minichromosome loss was significantly reduced in ku70Delta and lig4Delta non-homologous end joining (NHEJ) mutant backgrounds compared with wild type. Furthermore, roles for Ku70 and Lig4 were identified in suppressing DSB-induced chromosomal rearrangements associated with gene conversion. These findings are consistent with both competitive and cooperative interactions between components of the HR and NHEJ pathways.     

Topoisomerase I-dependent viability loss in saccharomyces cerevisiae mutants defective in both SUMO conjugation and DNA repair

Siz1 and Siz2/Nfi1 are the two Siz/PIAS SUMO E3 ligases in Saccharomyces cerevisiae. Here we show that siz1Delta siz2Delta mutants fail to grow in the absence of the homologous recombination pathway or the Fen1 ortholog RAD27. Remarkably, the growth defects of mutants such as siz1Delta siz2Delta rad52Delta are suppressed by mutations in TOP1, suggesting that these growth defects are caused by topoisomerase I activity. Other mutants that affect SUMO conjugation, including a ulp1 mutant and the nuclear pore mutants nup60Delta and nup133Delta, show similar top1-suppressible synthetic defects with DNA repair mutants, suggesting that these phenotypes also result from reduced SUMO conjugation. siz1Delta siz2Delta mutants also display TOP1-independent genome instability phenotypes, including increased mitotic recombination and elongated telomeres. We also show that SUMO conjugation, TOP1, and RAD27 have overlapping roles in telomere maintenance. Top1 is sumoylated, but Top1 does not appear to be the SUMO substrate involved in the synthetic growth defects. However, sumoylation of certain substrates, including Top1 itself and Tri1 (YMR233W), is enhanced in the absence of Top1 activity. Sumoylation is also required for growth of top1Delta cells. These results suggest that the SUMO pathway has a complex effect on genome stability that involves several mechanistically distinct processes.     

Short telomeres induce a DNA damage response in Saccharomyces cerevisiae

Telomerase-deficient Saccharomyces cerevisiae cells show a progressive decrease in telomere length. When grown for several days in log phase, the tlc1Delta cells initially display wild-type growth kinetics with subsequent loss of growth potential after which survivors are generated via RAD52-dependent homologous recombination. We found that chromosome loss in these telomerase-deficient cells only increased after a significant decline in growth potential of the culture. At earlier stages of growth, as the telomerase-deficient cells began to show loss of growth potential, the cells arrested in G2/M and showed RNR3 induction and Rad53p phosphorylation. These responses were dependent on RAD24 and MEC1, suggesting that short telomeres are recognized as DNA damage and signal G2/M arrest.     

DNA double-strand break repair pathways, chromosomal rearrangements and cancer

Chromosomal rearrangements, which can lead to oncogene activation and tumour suppressor loss, are a hallmark of cancer cells. Such outcomes can result from both the repair and misrepair of DNA ends, which arise from a variety of lesions including DNA double strand breaks (DSBs), collapsed replication forks and dysfunctional telomeres. Here we review the mechanisms by which non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways can both promote chromosomal rearrangements and also suppress them in response to such lesions, in accordance with their increasingly recognised tumour suppressor function. Further, we consider how chromosomal rearrangements, together with a modular approach towards understanding their etiology, may be exploited for cancer therapy.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.     

Length-dependent processing of telomeres in the absence of telomerase

In the absence of telomerase, telomeres progressively shorten with every round of DNA replication, leading to replicative senescence. In telomerase-deficient Saccharomyces cerevisiae, the shortest telomere triggers the onset of senescence by activating the DNA damage checkpoint and recruiting homologous recombination (HR) factors. Yet, the molecular structures that trigger this checkpoint and the mechanisms of repair have remained elusive. By tracking individual telomeres, we show that telomeres are subjected to different pathways depending on their length. We first demonstrate a progressive accumulation of subtelomeric single-stranded DNA (ssDNA) through 5′-3′ resection as telomeres shorten. Thus, exposure of subtelomeric ssDNA could be the signal for cell cycle arrest in senescence. Strikingly, early after loss of telomerase, HR counteracts subtelomeric ssDNA accumulation rather than elongates telomeres. We then asked whether replication repair pathways contribute to this mechanism. We uncovered that Rad5, a DNA helicase/Ubiquitin ligase of the error-free branch of the DNA damage tolerance (DDT) pathway, associates with native telomeres and cooperates with HR in senescent cells. We propose that DDT acts in a length-independent manner, whereas an HR-based repair using the sister chromatid as a template buffers precocious 5′-3′ resection at the shortest telomeres.     

A yeast-based genetic screening to identify human proteins that increase homologous recombination

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.     

Recruitment of Rad51 and Rad52 to Short Telomeres Triggers a Mec1-Mediated Hypersensitivity to Double-Stranded DNA Breaks in Senescent Budding Yeast

Telomere maintenance is required for chromosome stability, and telomeres are typically replicated by the action of telomerase. In both mammalian tumor and yeast cells that lack telomerase, telomeres are maintained by an alternative recombination mechanism. Here we demonstrated that the budding yeast Saccharomyces cerevisiae type I survivors derived from telomerase-deficient cells were hypersensitive to DNA damaging agents. Assays to track telomere lengths and drug sensitivity of telomerase-deficient cells from spore colonies to survivors suggested a correlation between telomere shortening and bleomycin sensitivity. Our genetic studies demonstrated that this sensitivity depends on Mec1, which signals checkpoint activation, leading to prolonged cell-cycle arrest in senescent budding yeasts. Moreover, we also observed that when cells equipped with short telomeres, recruitments of homologous recombination proteins, Rad51 and Rad52, were reduced at an HO-endonuclease-catalyzed double-strand break (DSB), while their associations were increased at chromosome ends. These results suggested that the sensitive phenotype may be attributed to the sequestration of repair proteins to compromised telomeres, thus limiting the repair capacity at bona fide DSB sites.     

Functional and genetic analysis of the Saccharomyces cerevisiae RNC1/TRM2: evidences for its involvement in DNA double-strand break repair

We previously isolated the RNC1/TRM2 gene and provided evidence that it encodes a protein with a possible role in DNA double strand break repair. RNC1 was independently re-isolated as the TRM2 gene encoding a methyl transferase involved in tRNA maturation. Here we show that Trm2p purified as a fusion protein displayed 5' --> 3' exonuclease activity on double-strand (ds) DNA, and endonuclease activity on single-strand (ss) DNA, properties characteristic of previously isolated endo-exonucleases. A variant of Trm2p, Trm2p(ctDelta76aa) lacking 76 amino acids at the C-terminus retained nuclease activities but not the methyl transferase activity. Both the native and the variant exhibited sensitivity to the endo-exonuclease inhibitor pentamidine. The Saccharomyces cerevisiae trm2(Delta232-1920nt) mutant (containing only the first 231 nucleotides of the TRM2 gene) displayed low sensitivity to methyl methane sulfonate (MMS) and suppressed the MMS sensitivity of rad52 mutants in trm2(Delta232-1920nt)rad52 double mutants. The deletion of KU80, in trm2(Delta232-1920nt) mutant background displayed higher MMS sensitivity supporting the view of the possible role of Trm2p in a competing repair pathway separate from NHEJ. In addition, trm2 exo1 double mutants were synergistically more sensitive to MMS and ionizing radiation than either of the single mutant suggesting that TRM2 and EXO1 can functionally complement each other. However, the C-terminal portion, required for its methyl transferase activity was found not important for DNA repair. These results propose an important role for TRM2 in DNA repair with a potential involvement of its nuclease function in homologous recombination based repair of DNA DSBs.     

Mutations in recombinational repair and in checkpoint control genes suppress the lethal combination of srs2Delta with other DNA repair genes in Saccharomyces cerevisiae

The SRS2 gene of Saccharomyces cerevisiae encodes a DNA helicase that is active in the postreplication repair pathway and homologous recombination. srs2 mutations are lethal in a rad54Delta background and cause poor growth or lethality in rdh54Delta, rad50Delta, mre11Delta, xrs2Delta, rad27Delta, sgs1Delta, and top3Delta backgrounds. Some of these genotypes are known to be defective in double-strand break repair. Many of these lethalities or poor growth can be suppressed by mutations in other genes in the DSB repair pathway, namely rad51, rad52, rad55, and rad57, suggesting that inhibition of recombination at a prior step prevents formation of a lethal intermediate. Lethality of the srs2Delta rad54Delta and srs2Delta rdh54Delta double mutants can also be rescued by mutations in the DNA damage checkpoint functions RAD9, RAD17, RAD24, and MEC3, indicating that the srs2 rad54 and srs2 rdh54 mutant combinations lead to an intermediate that is sensed by these checkpoint functions. When the checkpoints are intact the cells never reverse from the arrest, but loss of the checkpoints releases the arrest. However, cells do not achieve wild-type growth rates, suggesting that unrepaired damage is still present and may lead to chromosome loss.     

Mechanisms and regulation of DNA end resection

DNA double‐strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair these lesions can lead to genomic instability. DSBs can arise accidentally at unpredictable locations into the genome, but they are also normal intermediates in meiotic recombination. Moreover, the natural ends of linear chromosomes resemble DSBs. Although intrachromosomal DNA breaks are potent stimulators of the DNA damage response, the natural ends of linear chromosomes are packaged into protective structures called telomeres that suppress DNA repair/recombination activities. Although DSBs and telomeres are functionally different, they both undergo 5′–3′ nucleolytic degradation of DNA ends, a process known as resection. The resulting 3′‐single‐stranded DNA overhangs enable repair of DSBs by homologous recombination (HR), whereas they allow the action of telomerase at telomeres. The molecular activities required for DSB and telomere end resection are similar, indicating that the initial steps of HR and telomerase‐mediated elongation are related. Resection of both DSBs and telomeres must be tightly regulated in time and space to ensure genome stability and cell survival.     

Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.     

Ku suppresses formation of telomeric circles and alternative telomere lengthening in Arabidopsis

Telomeres in mammals and plants are protected by the terminal t loop structure, the formation of which parallels the first steps of intrachromatid homologous recombination (HR). Under some circumstances, cells can also utilize an HR-based mechanism (alternative lengthening of telomeres [ALT]) as a back-up pathway for telomere maintenance. We have found that the Ku70/80 heterodimer, a central nonhomologous end-joining DNA repair factor, inhibits engagement of ALT in Arabidopsis telomerase-negative cells. To further assess HR activities at telomeres, we have developed a sensitive assay for detecting extrachromosomal telomeric circles (t circles) that may arise from t loop resolution and aberrant HR. We show that Ku70/80 specifically inhibits circle formation at telomeres, but not at centromeric and rDNA repeats. Ku inactivation results in increased formation of t circles that represent approximately 4% of total telomeric DNA. However, telomeres in ku mutants are fully functional, indicating that telomerase efficiently heals ongoing terminal deletions arising from excision of the t circles.