Inactivation of Ku-mediated end joining suppresses mec1Delta lethality by depleting the ribonucleotide reductase inhibitor Sml1 through a pathway controlled by Tel1 kinase and the Mre11 complex

RAD53 and MEC1 are essential Saccharomyces cerevisiae genes required for the DNA replication and DNA damage checkpoint responses. Their lethality can be suppressed by increasing the intracellular pool of deoxynucleotide triphosphates. We report that deletion of YKU70 or YKU80 suppresses mec1Delta, but not rad53Delta, lethality. We show that suppression of mec1Delta lethality is not due to Ku--associated telomeric defects but rather results from the inability of Ku- cells to efficiently repair DNA double strand breaks by nonhomologous end joining. Consistent with these results, mec1Delta lethality is also suppressed by lif1Delta, which like yku70Delta and yku80Delta, prevents nonhomologous end joining. The viability of yku70Delta mec1Delta and yku80Delta mec1Delta cells depends on the ATM-related Tel1 kinase, the Mre11-Rad50-Xrs2 complex, and the DNA damage checkpoint protein Rad9. We further report that this Mec1-independent pathway converges with the Rad53/Dun1-regulated checkpoint kinase cascade and leads to the degradation of the ribonucleotide reductase inhibitor Sml1.     

The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis.

To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.     

Replication protein A is required for meiotic recombination in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, meiotic recombination is initiated by transient DNA double-stranded breaks (DSBs). These DSBs undergo a 5' --> 3' resection to produce 3' single-stranded DNA ends that serve to channel DSBs into the RAD52 recombinational repair pathway. In vitro studies strongly suggest that several proteins of this pathway--Rad51, Rad52, Rad54, Rad55, Rad57, and replication protein A (RPA)--play a role in the strand exchange reaction. Here, we report a study of the meiotic phenotypes conferred by two missense mutations affecting the largest subunit of RPA, which are localized in the protein interaction domain (rfa1-t11) and in the DNA-binding domain (rfa1-t48). We find that both mutant diploids exhibit reduced sporulation efficiency, very poor spore viability, and a 10- to 100-fold decrease in meiotic recombination. Physical analyses indicate that both mutants form normal levels of meiosis-specific DSBs and that the broken ends are processed into 3'-OH single-stranded tails, indicating that the RPA complex present in these rfa1 mutants is functional in the initial steps of meiotic recombination. However, the 5' ends of the broken fragments undergo extensive resection, similar to what is observed in rad51, rad52, rad55, and rad57 mutants, indicating that these RPA mutants are defective in the repair of the Spo11-dependent DSBs that initiate homologous recombination during meiosis.     

G1/S and G2/M Cyclin-Dependent Kinase Activities Commit Cells to Death in the Absence of the S-Phase Checkpoint

The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G₁ and mitosis. In fact, delaying either the G₁/S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions.     

Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.

We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.     

Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing

The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.     

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S‐phase checkpoint

The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1‐dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single‐strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1‐dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3′ ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.     

The roles of REV3 and RAD57 in double-strand-break-repair-induced mutagenesis of Saccharomyces cerevisiae

The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces approximately 75% of the BRIMs and approximately 90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol zeta during recombination.     

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G2 checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-D (null) mutant is defective in both S and G2 checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G2 checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1, and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G2 checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G2-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G2 arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.     

Srs2 DNA helicase is involved in checkpoint response and its regulation requires a functional Mec1-dependent pathway and Cdk1 activity

In Saccharomyces cerevisiae the rate of DNA replication is slowed down in response to DNA damage as a result of checkpoint activation, which is mediated by the Mec1 and Rad53 protein kinases. We found that the Srs2 DNA helicase, which is involved in DNA repair and recombination, is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner. DNA damage-induced Srs2 phosphorylation also requires the activity of the cyclin-dependent kinase Cdk1, suggesting that the checkpoint pathway might modulate Cdk1 activity in response to DNA damage. Moreover, srs2 mutants fail to activate Rad53 properly and to slow down DNA replication in response to intra-S DNA damage. The residual Rad53 activity observed in srs2 cells depends upon the checkpoint proteins Rad17 and Rad24. Moreover, DNA damage-induced lethality in rad17 mutants depends partially upon Srs2, suggesting that a functional Srs2 helicase causes accumulation of lethal events in a checkpoint-defective context. Altogether, our data implicate Srs2 in the Mec1 and Rad53 pathway and connect the checkpoint response to DNA repair and recombination.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.     

Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins.

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression was observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.     

Yeast Rad52 and Rad51 recombination proteins define a second pathway of DNA damage assessment in response to a single double-strand break

Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. We have found that both Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. We suggest that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p.     

RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates

DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.     

Analysis of the Tolerance to DNA Alkylating Damage in MEC1 and RAD53 Checkpoint Mutants of Saccharomyces cerevisiae

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.