The plant DNA polymerase theta is essential for the repair of replication-associated DNA damage

Safeguarding of genome integrity is a key process in all living organisms. Due to their sessile lifestyle, plants are particularly exposed to all kinds of stress conditions that could induce DNA damage. However, very few genes involved in the maintenance of genome integrity are indispensable to plants’ viability. One remarkable exception is the POLQ gene, which encodes DNA polymerase theta (Pol θ), a non-replicative polymerase involved in trans-lesion synthesis during DNA replication and double-strand break (DSB) repair. The Arabidopsis tebichi (teb) mutants, deficient in Pol θ, have been reported to display severe developmental defects, leading to the conclusion that Pol θ is required for normal plant development. However, this essential role of Pol θ in plants is challenged by contradictory reports regarding the phenotypic defects of teb mutants and the recent finding that rice (Oryza sativa) null mutants develop normally. Here we show that the phenotype of teb mutants is highly variable. Taking advantage of hypomorphic mutants for the replicative DNA polymerase epsilon, which display constitutive replicative stress, we show that Pol θ allows maintenance of meristem activity when DNA replication is partially compromised. Furthermore, we found that the phenotype of Pol θ mutants can be aggravated by modifying their growth conditions, suggesting that environmental conditions impact the basal level of replicative stress and providing evidence for a link between plants’ responses to adverse conditions and mechanisms involved in the maintenance of genome integrity.     

The fission yeast UVDR DNA repair pathway is inducible.

In addition to nucleotide excision repair (NER), the fission yeast Schizosaccharomyces pombe possesses a UV damage endonuclease (UVDE) for the excision of cyclobutane pyrimidine dimers and 6-4 pyrimidine pyrimidones. We have previously described UVDE as part of an alternative excision repair pathway, UVDR, for UV damage repair. The existence of two excision repair processes has long been postulated to exist in S.pombe, as NER-deficient mutants are still proficient in the excision of UV photoproducts. UVDE recognizes the phosphodiester bond immediately 5'of the UV photoproducts as the initiating event in this process. We show here that UVDE activity is inducible at both the level of uve1+ mRNA and UVDE enzyme activity. Further, we show that UVDE activity is regulated by the product of the rad12 gene.     

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.     

A homologue of the Rad18 postreplication repair gene is required for DNA damage responses throughout the fission yeast cell cycle

Cells activate DNA repair pathways and cell cycle checkpoints when they suffer damage to their genome. They also activate tolerance pathways that facilitate survival. In Escherichia coli, a mechanism known as postreplication repair (PRR) is used to bypass lesions that would otherwise present a physical block to DNA polymerase. PRR has also been proposed to occur in eukaryotic cells, although the partitioning of DNA synthesis to a discrete S-phase would suggest that it is only operative within a defined period of the cell cycle. Eukaryotic PRR has been most extensively studied in the budding yeast Saccharomyces cerevisiae. Two important genes for components of this repair pathway are RAD6, which encodes an ubiquitin-conjugating enzyme, and RAD18, which encodes a RING-finger protein and forms a heterodimer with Rad6p. Rad18p can also bind to DNA. We report here the identification of the Schizosaccharomyces pombe homologue of RAD18, which we have denoted rhp18. rhp18 mutants are hypersensitive to DNA-damaging agents, but show this hypersensitivity throughout the cell cycle. rhp18 mutants are characterised by a longer than usual DNA damage checkpoint arrest that is required for their residual viability following irradiation. Genetic analyses show that rhp18 controls a unique DNA damage repair/tolerance pathway that extends beyond the requirement to tolerate damage during S-phase, suggesting a broader definition of the function of this eukaryotic PRR protein.     

Assaying the DNA damage checkpoint in fission yeast

Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information. The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution. In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations. Fission yeast is an ideal system to investigate these pathways because there exist specific techniques that allow one to assay the fidelity of this DNA damage checkpoint pathway.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast

A checkpoint responding to DNA damage in G2 results in a delay in the onset of mitosis through inhibition of p34^cdc2 kinase activity via maintenance of inhibitory tyrosine phosphorylation. Genetic analyses of this checkpoint in fission yeast have identified single alleles of several genes, suggesting these screens are not yet saturating, and hence further genes await identification. To fully understand the complexity of this checkpoint it will be necessary to define all the genes involved. To this end we screened for new mutants defective in the ability to delay mitosis in the presence of DNA-damaging agents. Twenty-four mutants were isolated that were defective in UV-C and MMS-induced checkpoint delay. Amongst these mutants was an allele of cut5 that was also defective in the checkpoint responses. We show here, contrary to previous reports, that the UV-C induced checkpoint response is defective in cut5 mutants. Therefore, like all other checkpoint mutants, cut5 is required for G2 checkpoint arrest following DNA damage, regardless of the nature of the lesions involved. Received: 24 July 1998 / Accepted: 14 September 1998     

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.     

Gene HI1472 of Haemophilus influenzae Rd is a novel gene involved in DNA repair

A chimeric plasmid, pJPuvr4, consists of a 16.7 kbp Haemophilus influenzae Rd chromosomal DNA insert at the EcoRI site of vector pJ1-8. This plasmid complements the UV and gamma ray sensitivity of the mutant strain MBH4. This plasmid carries the wild type allele of gene uvr4 which was localised to a 3.8 kbp DraI fragment, with an internal EcoRI site. Partial sequencing of the gene and its alignment with the published genome sequence of H. influenzae Rd revealed uvr4 to be HI1472. HI1472 is a putatively identified open reading frame (ORF), which has been assigned no function so far. The partial sequence did show nt database match with 3D exon of N cadherin gene of homosepians and moaA gene of H. influenzae. Cadherins are involved in cell adhesion, cell to cell contact and morphogenesis in homosepians and moaA gene codes for molybdenum biosynthesis subunitA. This report implicates HI1472 of Haemophilus influenzae Rd in DNA repair. Nucleotide sequence obtained for the gene uvr4 was compared with the published sequence of gene HI1472. A wild type strain variation was observed at the 592nd nucleotide position corresponding to a change from aspartic acid to threonine.     

Five novel elements involved in the regulation of mitosis in fission yeast

Summary Five new elements of the mitotic control in the fission yeast Schizosaccharomyces pombe were isolated from gene libraries as multicopy suppressors of the conditional lethal phenotype of win1-1 weel ^ts cdc25^ts triple mutant strains. These genes were designated wisl ⁺ –wis5⁺for win suppressing, and do not correspond to winl ⁺ or any of the previously characterised mitotic control genes. None of the wis genes is capable of suppressing the cdc phenotype of cdc25 ^ts strains, suggesting that their effect is not simply to reverse the effect of loss of cdc25 function. wisl ⁺ has been previously reported to encode a putative serine/threonine protein kinase that acts as a dosage-dependent inducer of mitosis. wis4 ⁺ appears to be a specific suppressor of the winl-1 mutation. wis2 ⁺ and wis3 ⁺ are capable of suppressing a wide range of cdc phenotypes arising from the combination of various mutations with wee1 ^ts and cdc25 ^ts, suggesting that the wis2 ⁺ and wis3 ⁺ products may interact with elements central to the mitotic control.     

The novel gene LRP15 is regulated by DNA methylation and confers increased efficiency of DNA repair of ultraviolet-induced DNA damage

LRP15 is a novel gene cloned from lymphocytic cells, and its function is still unknown. Bioinformatic data showed that LRP15 might be regulated by DNA methylation and had an important role in DNA repair. In this study, we investigate whether the expression of LRP15 is regulated by DNA methylation, and whether overexpression of LRP15 increases efficiency of DNA repair of UV-induced DNA damage in HeLa cells. The results showed (1) the promoter of LRP15 was hypermethylated in HeLa cells, resulting a silence of its expression. Gene expression was restored by a demethylating agent, 5-aza-2'-deoxycytidine, but not by a histone deacetylase inhibitor, trichostatin A; (2) overexpression of LRP15 inhibited HeLa cell proliferation, and the numbers of cells in the G2/M phase of the cell cycle in cells transfected with LRP15 increased about 10% compared with controls; (3) cyclin B1 level was much lower in cells overexpressing LRP15 than in control cells; and (4) after exposure to UV radiation, the LRP15-positive cells showed shorter comet tails compared with the LRP15-negative cells. From these results we conclude that the expression of LRP15 is controlled by methylation in its promoter in HeLa cells, and LRP15 confers resistance to UV damage and accelerates the DNA repair rate.     

DNA repair in the fission yeast, Schizosaccharomyces pombe

Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV and/or γ-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.     

The polyubiquitin gene is essential for meiosis in fission yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.     

Nuclear exclusion of Cdc25 is not required for the DNA damage checkpoint in fission yeast

Maintenance of genome integrity requires a checkpoint that restrains mitosis in response to DNA damage [1]. This checkpoint is enforced by Chk1, a protein kinase that targets Cdc25 [2--7]. Phosphorylated Cdc25 associates with 14-3-3 proteins, which appear to occlude a nuclear localization signal (NLS) and thereby inhibit Cdc25 nuclear import [6, 8--14]. Proficient checkpoint arrest is thought to require Cdc25 nuclear exclusion, although definitive evidence for this model is lacking. We have tested this hypothesis in fission yeast. We show that elimination of an NLS in Cdc25 causes Cdc25 nuclear exclusion and a mitotic delay, as predicted by the model. Attachment of an exogenous NLS forces nuclear inclusion of Cdc25 in damaged cells. However, forced nuclear localization of Cdc25 fails to override the damage checkpoint. Thus, nuclear exclusion of Cdc25 is unnecessary for checkpoint enforcement. We propose that direct inhibition of Cdc25 phosphatase activity by Chk1, as demonstrated in vitro with fission yeast and human Chk1 [15, 16], is sufficient for proficient checkpoint regulation of Cdc25 and may be the primary mechanism of checkpoint enforcement in fission yeast.     

Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells

Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.