RecN spatially and temporally controls RecA-mediated repair of DNA double-strand breaks.

RecN, a bacterial structural maintenance of chromosomes-like protein, plays an important role in maintaining genomic integrity by facilitating the repair of DNA double-strand breaks (DSBs). However, how RecN-dependent chromosome dynamics are integrated with DSB repair remains unclear. Here, we investigated the dynamics of RecN in response to DNA damage by inducing RecN from the PBAD promoter at different time points. We found that mitomycin C (MMC)-treated ΔrecN cells exhibited nucleoid fragmentation and reduced cell survival; however, when RecN was induced with arabinose in MMC-exposed ΔrecN cells, it increased a level of cell viability to similar extent as WT cells. Furthermore, in MMC-treated ΔrecN cells, arabinose-induced RecN colocalized with RecA in nucleoid gaps between fragmented nucleoids and restored normal nucleoid structures. These results suggest that the aberrant nucleoid structures observed in MMC-treated ΔrecN cells do not represent catastrophic chromosome disruption but rather an interruption of the RecA-mediated process. Thus, RecN can resume DSB repair by stimulating RecA-mediated homologous recombination, even when chromosome integrity is compromised. Our data demonstrate that RecA-mediated presynapsis and synapsis are spatiotemporally separable, wherein RecN is involved in facilitating both processes presumably by orchestrating the dynamics of both RecA and chromosomes, highlighting the essential role of RecN in the repair of DSBs.

Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present.IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

UNAGI: an automated pipeline for nanopore full-length cDNA sequencing uncovers novel transcripts and isoforms in yeast.

Sequencing the entire RNA molecule leads to a better understanding of the transcriptome architecture. SMARTer (Switching Mechanism at 5'-End of RNA Template) is a technology aimed at generating full-length cDNA from low amounts of mRNA for sequencing by short-read sequencers such as those from Illumina. However, short read sequencing such as Illumina technology includes fragmentation that results in bias and information loss. Here, we built a pipeline, UNAGI or UNAnnotated Gene Identifier, to process long reads obtained with nanopore sequencing and compared this pipeline with the standard Illumina pipeline by studying the Saccharomyces cerevisiae transcriptome in full-length cDNA samples generated from two different biological samples: haploid and diploid cells. Additionally, we processed the long reads with another long read tool, FLAIR. Our strand-aware method revealed significant differential gene expression that was masked in Illumina data by antisense transcripts. Our pipeline, UNAGI, outperformed the Illumina pipeline and FLAIR in transcript reconstruction (sensitivity and specificity of 80% and 40% vs. 18% and 34% and 79% and 32%, respectively). Moreover, UNAGI discovered 3877 unannotated transcripts including 1282 intergenic transcripts while the Illumina pipeline discovered only 238 unannotated transcripts. For isoforms profiling, UNAGI also outperformed the Illumina pipeline and FLAIR in terms of sensitivity (91% vs. 82% and 63%, respectively). But the low accuracy of nanopore sequencing led to a closer gap in terms of specificity with Illumina pipeline (70% vs. 63%) and to a huge gap with FLAIR (70% vs 0.02%).

Srs2 and Mus81-Mms4 Prevent Accumulation of Toxic Inter-Homolog Recombination Intermediates.

Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81-Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability.

RAD6-RAD18-RAD5-pathway-dependent tolerance to chronic low-dose ultraviolet light.

In nature, organisms are exposed to chronic low-dose ultraviolet light (CLUV) as opposed to the acute high doses common to laboratory experiments. Analysis of the cellular response to acute high-dose exposure has delineated the importance of direct DNA repair by the nucleotide excision repair pathway and for checkpoint-induced cell cycle arrest in promoting cell survival. Here we examine the response of yeast cells to CLUV and identify a key role for the RAD6-RAD18-RAD5 error-free postreplication repair (RAD6 error-free PRR) pathway in promoting cell growth and survival. We show that loss of the RAD6 error-free PRR pathway results in DNA-damage-checkpoint-induced G2 arrest in CLUV-exposed cells, whereas wild-type and nucleotide-excision-repair-deficient cells are largely unaffected. Cell cycle arrest in the absence of the RAD6 error-free PRR pathway was not caused by a repair defect or by the accumulation of ultraviolet-induced photoproducts. Notably, we observed increased replication protein A (RPA)- and Rad52-yellow fluorescent protein foci in the CLUV-exposed rad18Delta cells and demonstrated that Rad52-mediated homologous recombination is required for the viability of the rad18Delta cells after release from CLUV-induced G2 arrest. These and other data presented suggest that, in response to environmental levels of ultraviolet exposure, the RAD6 error-free PRR pathway promotes replication of damaged templates without the generation of extensive single-stranded DNA regions. Thus, the error-free PRR pathway is specifically important during chronic low-dose ultraviolet exposure to prevent counter-productive DNA checkpoint activation and allow cells to proliferate normally.

RecA protein recruits structural maintenance of chromosomes (SMC)-like RecN protein to DNA double-strand breaks.

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA(Q300R), is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA(Q300R) fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN(K35A) binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

Chronic low-dose ultraviolet-induced mutagenesis in nucleotide excision repair-deficient cells.

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14Δ NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14Δ cells have a marked increase in CLUV-induced mutations, most of which are C→T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C→T mutations in rad14Δ cells are dependent on translesion synthesis (TLS) DNA polymerase η, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Polη in the induction of C→T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Polη can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2-E3 pairs.

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.

N-terminal acetyltransferase NatB regulates Rad51-dependent repair of double-strand breaks in Saccharomyces cerevisiae.

Homologous recombination (HR) is a highly accurate mechanism for repairing DNA double-strand breaks (DSBs) that arise from various genotoxic insults and blocked replication forks. Defects in HR and unscheduled HR can interfere with other cellular processes such as DNA replication and chromosome segregation, leading to genome instability and cell death. Therefore, the HR process has to be tightly controlled. Protein N-terminal acetylation is one of the most common modifications in eukaryotic organisms. Studies in budding yeast implicate a role for NatB acetyltransferase in HR repair, but precisely how this modification regulates HR repair and genome integrity is unknown. In this study, we show that cells lacking NatB, a dimeric complex composed of Nat3 and Mdm2, are sensitive to the DNA alkylating agent methyl methanesulfonate (MMS), and that overexpression of Rad51 suppresses the MMS sensitivity of nat3Δ cells. Nat3-deficient cells have increased levels of Rad52-yellow fluorescent protein foci and fail to repair DSBs after release from MMS exposure. We also found that Nat3 is required for HR-dependent gene conversion and gene targeting. Importantly, we observed that nat3Δ mutation partially suppressed MMS sensitivity in srs2Δ cells and the synthetic sickness of srs2Δ sgs1Δ cells. Altogether, our results indicate that NatB functions upstream of Srs2 to activate the Rad51-dependent HR pathway for DSB repair.

Diploid-associated adaptation to chronic low-dose UV irradiation requires homologous recombination in Saccharomyces cerevisiae.

Ultraviolet-induced DNA lesions impede DNA replication and transcription and are therefore a potential source of genome instability. Here, we performed serial transfer experiments on nucleotide excision repair-deficient (rad14Δ) yeast cells in the presence of chronic low-dose ultraviolet irradiation, focusing on the mechanisms underlying adaptive responses to chronic low-dose ultraviolet irradiation. Our results show that the entire haploid rad14Δ population rapidly becomes diploid during chronic low-dose ultraviolet exposure, and the evolved diploid rad14Δ cells were more chronic low-dose ultraviolet-resistant than haploid cells. Strikingly, single-stranded DNA, but not pyrimidine dimer, accumulation is associated with diploid-dependent fitness in response to chronic low-dose ultraviolet stress, suggesting that efficient repair of single-stranded DNA tracts is beneficial for chronic low-dose ultraviolet tolerance. Consistent with this hypothesis, homologous recombination is essential for the rapid evolutionary adaptation of diploidy, and rad14Δ cells lacking Rad51 recombinase, a key player in homologous recombination, exhibited abnormal cell morphology characterized by multiple RPA-yellow fluorescent protein foci after chronic low-dose ultraviolet exposure. Furthermore, interhomolog recombination is increased in chronic low-dose ultraviolet-exposed rad14Δ diploids, which causes frequent loss of heterozygosity. Thus, our results highlight the importance of homologous recombination in the survival and genomic stability of cells with unrepaired lesions.

A Conserved Histone H3-H4 Interface Regulates DNA Damage Tolerance and Homologous Recombination during the Recovery from Replication Stress.

In eukaryotes, genomic DNA is packaged into nucleosomes, which are the basal components coordinating both the structures and functions of chromatin. In this study, we screened a collection of mutations for histone H3/H4 mutants in Saccharomyces cerevisiae that affect the DNA damage sensitivity of DNA damage tolerance (DDT)-deficient cells. We identified a class of histone H3/H4 mutations that suppress methyl methanesulfonate (MMS) sensitivity of DDT-deficient cells (referred to here as the histone SDD mutations), which likely cluster on a specific H3-H4 interface of the nucleosomes. The histone SDD mutations did not suppress the MMS sensitivity of DDT-deficient cells in the absence of Rad51, indicating that homologous recombination (HR) is responsible for DNA damage resistance. Furthermore, the histone SDD mutants showed reduced levels of PCNA ubiquitination after exposure to MMS or UV irradiation, consistent with decreased MMS-induced mutagenesis relative to that of wild-type cells. We also found that histone SDD mutants lacking the INO80 chromatin remodeler impair HR-dependent recovery from MMS-induced replication arrest, resulting in defective S-phase progression and increased Rad52 foci. Taken together, our data provide novel insights into nucleosome functions, which link INO80-dependent chromatin remodeling to the regulation of DDT and HR during the recovery from replication blockage.

Monitoring of DNA Replication and DNA Double-Strand Breaks in Saccharomyces cerevisiae by Pulsed-Field Gel Electrophoresis (PFGE).

Separating DNA fragments using standard agarose gel electrophoresis is based on the capacity of negatively charged DNA molecules to move through the agarose gel matrix toward the positive electrode. Pulsed-field gel electrophoresis (PFGE) is an agarose gel electrophoresis technique that enables the separation of DNA molecules at a megabase scale, making the direct genomic analysis of large DNA molecules possible. For instance, 16 chromosomes (size range; 0.2-2.2 Mb) in Saccharomyces cerevisiae, whose karyotype cannot be easily observed with a microscope, can be directly separated on agarose gel. PFGE is also a powerful analytical tool for chromosomal mapping and genome structure analysis in bacterial and mammalian cells. In this chapter, we will describe the preparation of intact yeast chromosomal DNA for PFGE and general PFGE procedures and will introduce a PFGE method to monitor the DNA replication fork progression and DNA double-strand breaks (DSBs).

A SUMO-like domain protein, Esc2, is required for genome integrity and sister chromatid cohesion in Saccharomyces cerevisiae.

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2Delta cells are modestly sensitive to hydroxyurea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2Delta rrm3Delta cells also have a severe growth defect and accumulate DNA damage in late S/G2. In contrast, esc2Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3Delta cells.

Rhp51-dependent recombination intermediates that do not generate checkpoint signal are accumulated in Schizosaccharomyces pombe rad60 and smc5/6 mutants after release from replication arrest.

The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.

Functional and physical interaction of yeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance.

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli.

Bacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified Escherichia coli RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair.

BACKGROUND: DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. RESULTS: To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. CONCLUSION: These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Cyclin-dependent kinase modulates budding yeast Rad5 stability during cell cycle.

The DNA damage tolerance (DDT) pathway facilitates the bypass of the fork-blocking lesions without removing them through either translesion DNA synthesis or error-free damage bypass mechanism. The Saccharomyces cerevisiae Rad5 is a multi-functional protein involved in the error-free branch of the DDT pathway, and its protein level periodically fluctuates through the cell cycle; however, the mechanistic basis and functional importance of the Rad5 level for the cell cycle regulation remain unclear. Here, we show that Rad5 is predominantly phosphorylated on serine 130 (S130) during S/G2 phase and that this modification depends on the cyclin-dependent kinase Cdc28/CDK1. We also show that the phosphorylated Rad5 species at S130 exhibit a relatively short half-life compared with non-phosphorylated Rad5 moiety, and that the Rad5 protein is partially stabilized in phosphorylation-defective rad5 S130A cells. Importantly, the elimination of this modification results in a defective cell-cycle dependent Rad5 oscillation pattern. Together, our results demonstrate that CDK1 modulates Rad5 stability by phosphorylation during the cell cycle, suggesting a crosstalk between the phosphorylation and degradation of Rad5.