Intrinsic 5'-deoxyribose-5-phosphate lyase activity in Saccharomyces cerevisiae Trf4 protein with a possible role in base excision DNA repair

In Saccharomyces cerevisiae, the base excision DNA repair (BER) pathway has been thought to involve only a multinucleotide (long-patch) mechanism (LP-BER), in contrast to most known cases that include a major single-nucleotide pathway (SN-BER). The key step in mammalian SN-BER, removal of the 5'-terminal abasic residue generated by AP endonuclease incision, is effected by DNA polymerase beta (Polbeta). Computational analysis indicates that yeast Trf4 protein, with roles in sister chromatin cohesion and RNA quality control, is a new member of the X family of DNA polymerases that includes Polbeta. Previous studies of yeast trf4Delta mutants revealed hypersensitivity to methylmethane sulfonate (MMS) but not UV light, a characteristic of BER mutants in other organisms. We found that, like mammalian Polbeta, Trf4 is able to form a Schiff base intermediate with a 5'-deoxyribose-5-phosphate substrate and to excise the abasic residue through a dRP lyase activity. Also like Polbeta, Trf4 forms stable cross-links in vitro to 5'-incised 2-deoxyribonolactone residues in DNA. We determined the sensitivity to MMS of strains with a trf4Delta mutation in a rad27Delta background, in an AP lyase-deficient background (ogg1 ntg1 ntg2), or in a pol4Delta background. Only a RAD27 genetic interaction was detected: there was higher sensitivity for strains mutated in both TRF4 and RAD27 than either single mutant, and overexpression of Trf4 in a rad27Delta background partially suppressed MMS sensitivity. The data strongly suggest a role for Trf4 in a pathway parallel to the Rad27-dependent LP-BER in yeast. Finally, we demonstrate that Trf5 significantly affects MMS sensitivity and thus probably BER efficiency in cells expressing either wild-type Trf4 or a C-terminus-deleted form.     

MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.     

The Saccharomyces cerevisiae DNA damage checkpoint is required for efficient repair of double strand breaks by non-homologous end joining

In this work we report that the Saccharomyces cerevisiae RAD9, RAD24, RAD17, MEC1, MEC3 and RAD53 checkpoint genes are required for efficient non-homologous end joining (NHEJ). RAD9 and RAD24 function additionally in this process. Defective NHEJ in rad9Delta-rad24Delta, but not yku80Delta cells, is only partially rescued by imposing G1 or G2/M delays. Thus, checkpoint functions other than transient cell cycle delays may be required for normal levels of NHEJ. Epistasis analysis also indicated that YKU80 and RAD9/RAD24 function in the same pathway for repair of lesions caused by MMS and gamma-irradiation. Unlike NHEJ, the checkpoint pathway is not required for efficient site-specific integration of plasmid DNA into the yeast genome, which is RAD52-dependent, but RAD51-independent.     

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.     

Functional conservation of the yeast and Arabidopsis RAD54-like genes

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance severalfold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54Delta mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.     

Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.

Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.     

酵母DNA修复基因RAD16温度敏感突变株的分离及人类互补基因的鉴定

用PCR介导的基因重组法敲除酵母RAD16基因, 用羟胺处理酵母RAD16基因表达质粒, 获得RAD16基因突变库,并将其转化RAD16基因敲除的酵母细胞. 用平皿复制法获得温度敏感突变株 rad16-ts2,经互补试验证实,该突变表型为RAD16基因突变所致.将人cDNA表达文库转化rad16-ts2后筛选温度敏感拯救(rescue)表型, 回收拯救表型酵母细胞中的质粒. 测序结果表明,所获得的人cDNA克隆为HLTF/Zbu1/SMACA3基因.比较分析显示,人类该基因编码蛋白质氨基酸序列与酵母Rad16的同一性为32%,相似性则达50%.人HLTF/Zbu1/MACA3基因在HeLa细胞中过表达,可显著抑制过氧化氢损伤和UV照射诱导的细胞凋亡.     

Sts1 can overcome the loss of Rad23 and Rpn10 and represents a novel regulator of the ubiquitin/proteasome pathway

A rad23Delta rpn10Delta double mutant accumulates multi-Ub proteins, is deficient in proteolysis, and displays sensitivity to drugs that generate damaged proteins. Overexpression of Sts1 restored normal growth in rad23Delta rpn10Delta but did not overcome the DNA repair defect of rad23Delta. To understand the nature of Sts1 suppression, we characterized sts1-2, a temperature-sensitive mutant. We determined that sts1-2 was sensitive to translation inhibitors, accumulated high levels of multi-Ub proteins, and caused stabilization of proteolytic substrates. Additionally, ubiquitinated proteins that were detected in proteasomes were inefficiently cleared in sts1-2. Despite these proteolytic defects, overall proteasome activity was increased in sts1-2. We propose that Sts1 is a new regulatory factor in the ubiquitin/proteasome pathway that controls the turnover of proteasome substrates.     

Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast

The yeast RAD27 gene encodes a functional homolog of the mammalian FEN1 protein, a structure-specific endo/exonuclease which plays an important role in DNA replication and repair. Previous genetic interaction studies, including a synthetic genetic array (SGA) analysis, showed that the survival of rad27Delta cells requires several DNA metabolic processes, in particular those mediated by all members of the Rad52-dependent recombinational repair pathway. Here, we report the results of our SGA analysis of the collection of non-essential yeast genes against the rad27Delta mutation, which resulted in the identification of a novel synthetic lethal interaction conferred by mutations affecting the Nup84 nuclear pore subcomplex (nup133Delta, nup120Delta and nup84Delta). Additional screens showed that all Rad52 group genes are required for the survival of the nup133Delta and nup120Delta mutants, which are defective in nuclear pore distribution and mRNA export, but not of the nup133DeltaN mutant, which is solely defective in pore distribution. This requirement for the DNA double-strand break (DSB) repair pathway is consistent with the observation that, like rad27Delta, the nup133Delta, nup120Delta and nup84Delta mutants are sensitive to methyl methanesulfonate (MMS). Furthermore, nup133Delta cells exhibit an increased number of spontaneous DNA repair foci containing Rad52. Altogether, these data suggest that the pathological interactions between the rad27Delta and specific nupDelta mutations result from the accumulation of unrepaired DNA damages.     

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.     

Chl12 (Ctf18) forms a novel replication factor C-related complex and functions redundantly with Rad24 in the DNA replication checkpoint pathway

RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. The rad24Delta mutation enhances the defect of rfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint. CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Delta nor rad24Delta single mutants are defective, chl12Delta rad24Delta double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.     

Genetic requirements for RAD51- and RAD54-independent break-induced replication repair of a chromosomal double-strand break

Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence of RAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as with rad51Delta cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1 (RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Delta rad50Delta, rad51Delta rad59Delta, and rad54Delta tid1Delta. These results demonstrate that there is a RAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumably MRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.     

Functional Validation of Rare Human Genetic Variants Involved in Homologous Recombination Using Saccharomyces cerevisiae

Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.     

RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination.

The RAD1 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of damaged DNA. In this paper, we report our observations on the effect of the RAD1 gene on genetic recombination. Mitotic intrachromosomal and interchromosomal recombination in RAD+, rad1, rad52, and other rad mutant strains was examined. The rad1 deletion mutation and some rad1 point mutations reduced the frequency of intrachromosomal recombination of a his3 duplication, in which one his3 allele is deleted at the 3' end while the other his3 allele is deleted at the 5' end. Mutations in the other excision repair genes, RAD2, RAD3, and RAD4, did not lower recombination frequencies in the his3 duplication. As expected, recombination between the his3 deletion alleles in the duplication was reduced in the rad52 mutant. The frequency of HIS3+ recombinants fell synergistically in the rad1 rad52 double mutant, indicating that the RAD1 and RAD52 genes affect this recombination via different pathways. In contrast to the effect of mutations in the RAD52 gene, mutations in the RAD1 gene did not lower intrachromosomal and interchromosomal recombination between heteroalleles that carry point mutations rather than partial deletions; however, the rad1 delta mutation did lower the frequency of integration of linear plasmids and DNA fragments into homologous genomic sequences. We suggest that RAD1 plays a role in recombination after the formation of the recombinogenic substrate.     

Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.     

A core activity associated with the N terminus of the yeast RAD52 protein is revealed by RAD51 overexpression suppression of C-terminal rad52 truncation alleles.

C-terminal rad52 truncation and internal deletion mutants were characterized for their ability to repair MMS-induced double-strand breaks and to produce viable spores during meiosis. The rad52-Delta251 allele, encoding the N-terminal 251 amino acids of the predicted 504-amino-acid polypeptide, supports partial activity for both functions. Furthermore, RAD51 overexpression completely suppresses the MMS sensitivity of a rad52-Delta251 mutant. The absence of the C terminus in the truncated protein makes it likely that suppression occurs by bypassing the C-terminal functions of Rad52p. RAD51 overexpression does not suppress the low level of spore viability that the rad52-Delta251 allele causes and only partially suppresses the defect in rad52 alleles encoding the N-terminal 292 or 327 amino acids. The results of this study also show that intragenic complementation between rad52 alleles is governed by a complex relationship that depends heavily on the two alleles involved and their relative dosage. In heteroallelic rad52 diploids, the rad52-Delta251 allele does not complement rad52 missense mutations altering residues 61 or 64 in the N terminus. However, complementation is achieved with each of these missense alleles when the rad52-Delta251 allele is overexpressed. Complementation also occurs between rad52-Delta327 and an internal deletion allele missing residues 210 through 327. We suggest that the first 251 amino acids of Rad52p constitute a core domain that provides critical RAD52 activities.     

Rfc5, in cooperation with rad24, controls DNA damage checkpoints throughout the cell cycle in Saccharomyces cerevisiae

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24Delta mutants are defective in all the G(1)-, S-, and G(2)/M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine(115) residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G(1)- and G(2)/M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24(K115R) fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G(1)-, S- and G(2)/M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.     

Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.     

Characterization of RAD52 homologs in the fission yeast Schizosaccharomyces pombe

The RAD52 gene of Saccharomyces cerevisiae is essential for repair of DNA double-strand breaks (DSBs) by homologous recombination. Inactivation of this gene confers hypersensitivity to DSB-inducing agents and defects in most forms of recombination. The rad22+ gene in Schizosaccharomyces pombe (here referred to as rad22A+) has been characterized as a homolog of RAD52 in fission yeast. Here, we report the identification of a second RAD52 homolog in Schizosaccharomyces pombe, called rad22B+. The amino acid sequences of Rad22A and Rad22B show significant conservation (38% identity). Deletion mutants of respectively, rad22A and rad22B, show different phenotypes with respect to sensitivity to X-rays and the ability to perform homologous recombination as measured by the integration of plasmid DNA. Inactivation of rad22A+ leads to a severe sensitivity to X-rays and a strong decrease in recombination (13-fold), while the rad22B mutation does not result in a decrease in homologous recombination or a change in radiation sensitivity. In a rad22A-rad22B double mutant the radiation sensitivity is further enhanced in comparison with the rad22A single mutant. Overexpression of the rad22B+ gene results in partial suppression of the DNA repair defects of the rad22A mutant strain. Meiotic recombination and spore viability are only slightly affected in either single mutant, but outgrowth of viable spores is almost 31-fold reduced in the rad22A-rad22B double mutant. The results obtained imply a crucial role for rad22A+ in repair and recombination in vegetative cells just like RAD52 in S. cerevisiae. The rad22B+ gene presumably has an auxiliary role in the repair of DSBs. The drastic reduced spore viability in the double mutant suggests that meiosis in S. pombe is dependent on the presence of either rad22A+ or rad22B+.