Sister Chromatid Recombination Research Articles

A Role for the Interactions between Polδ and PCNA Revealed by Analysis of pol3-01 Yeast Mutants.

Several DNA polymerases participate in DNA synthesis during genome replication and DNA repair. PCNA, a homotrimeric ring, acts as a processivity factor for DNA polymerases. PCNA also acts as a "landing pad" for proteins that interact with chromatin and DNA at the moving fork. The interaction between PCNA and polymerase delta (Polδ) is mediated by PIPs (PCNA-interacting peptides), in particular the one on Pol32, a regulatory subunit of Polδ. Here, we demonstrate that pol3-01, an exonuclease mutant of Polδ's catalytic subunit, exhibits a weak interaction with Pol30 compared to the WT DNA polymerase. The weak interaction activates DNA bypass pathways, leading to increased mutagenesis and sister chromatid recombination. Strengthening pol3-01's weak interaction with PCNA suppresses most of the phenotypes. Our results are consistent with a model in which Pol3-01 tends to detach from the chromatin, allowing an easier replacement of Polδ by the trans-lesion synthesis polymerase Zeta (Polz), thus leading to the increased mutagenic phenotype.

Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes

BackgroundCohesin is a chromosome-associated SMC–kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes.ResultsThrough high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein–condensin complexes from the prophase onward.ConclusionsOur findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Genome Profiling for Aflatoxin B1 Resistance in Saccharomyces cerevisiae Reveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-Associated DNA Damage

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 μM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

Loss of CBX2 induces genome instability and senescence-associated chromosomal rearrangements

The polycomb group protein CBX2 is an important epigenetic reader involved in cell proliferation and differentiation. While CBX2 overexpression occurs in a wide range of human tumors, targeted deletion results in homeotic transformation, proliferative defects, and premature senescence. However, its cellular function(s) and whether it plays a role in maintenance of genome stability remain to be determined. Here, we demonstrate that loss of CBX2 in mouse fibroblasts induces abnormal large-scale chromatin structure and chromosome instability. Integrative transcriptome analysis and ATAC-seq revealed a significant dysregulation of transcripts involved in DNA repair, chromocenter formation, and tumorigenesis in addition to changes in chromatin accessibility of genes involved in lateral sclerosis, basal transcription factors, and folate metabolism. Notably, Cbx2-/- cells exhibit prominent decondensation of satellite DNA sequences at metaphase and increased sister chromatid recombination events leading to rampant chromosome instability. The presence of extensive centromere and telomere defects suggests a prominent role for CBX2 in heterochromatin homeostasis and the regulation of nuclear architecture.

The nuclear pore complex prevents sister chromatid recombination during replicative senescence

The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to nuclear pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats. Strikingly, telomerase negative cells bypass senescence when expressing this Nup1 modification by maintaining a minimal telomere length compatible with proliferation through rampant unequal exchanges between sister chromatids. We further report that a Nup1 mutant lacking 36 C-terminal residues recapitulates the phenotypes of the Nup1-LexA fusion indicating a direct role of Nup1 in the relocation of stalled forks to NPCs and restriction of error-prone recombination between repeated sequences. Our results reveal a new mode of telomere maintenance that could shed light on how 20% of cancer cells are maintained without telomerase or ALT.

Distinct roles for S. cerevisiae H2A copies in recombination and repeat stability, with a role for H2A.1 threonine 126.

CAG/CTG trinuncleotide repeats are fragile sequences that when expanded form DNA secondary structures and cause human disease. We evaluated CAG/CTG repeat stability and repair outcomes in histone H2 mutants in S. cerevisiae. Although the two copies of H2A are nearly identical in amino acid sequence, CAG repeat stability depends on H2A copy 1 (H2A.1) but not copy 2 (H2A.2). H2A.1 promotes high-fidelity homologous recombination, sister chromatid recombination (SCR), and break-induced replication whereas H2A.2 does not share these functions. Both decreased SCR and the increase in CAG expansions were due to the unique Thr126 residue in H2A.1 and hta1Δ or hta1-T126A mutants were epistatic to deletion of the Polδ subunit Pol32, suggesting a role for H2A.1 in D-loop extension. We conclude that H2A.1 plays a greater repair-specific role compared to H2A.2 and may be a first step towards evolution of a repair-specific function for H2AX compared to H2A in mammalian cells.

Rpd3L and Hda1 histone deacetylases facilitate repair of broken forks by promoting sister chromatid cohesion

Genome stability involves accurate replication and DNA repair. Broken replication forks, such as those encountering a nick, lead to double strand breaks (DSBs), which are preferentially repaired by sister-chromatid recombination (SCR). To decipher the role of chromatin in eukaryotic DSB repair, here we analyze a collection of yeast chromatin-modifying mutants using a previously developed system for the molecular analysis of repair of replication-born DSBs by SCR based on a mini-HO site. We confirm the candidates through FLP-based systems based on a mutated version of the FLP flipase that causes nicks on either the leading or lagging DNA strands. We demonstrate that Rpd3L and Hda1 histone deacetylase (HDAC) complexes contribute to the repair of replication-born DSBs by facilitating cohesin loading, with no effect on other types of homology-dependent repair, thus preventing genome instability. We conclude that histone deacetylation favors general sister chromatid cohesion as a necessary step in SCR.

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells.

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hyper-sensitivity to mitomycin C and olaparib, with the weakest phenotypes observed in RAD51B-deficient cells. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

Ribosomal RNA gene repeats associate with the nuclear pore complex for maintenance after DNA damage

The ribosomal RNA genes (rDNA) comprise a highly repetitive gene cluster. The copy number of genes at this locus can readily change and is therefore one of the most unstable regions of the genome. DNA damage in rDNA occurs after binding of the replication fork blocking protein Fob1 in S phase, which triggers unequal sister chromatid recombination. However, the precise mechanisms by which such DNA double-strand breaks (DSBs) are repaired is not well understood. Here, we demonstrate that the conserved protein kinase Tel1 maintains rDNA stability after replication fork arrest. We show that rDNA associates with nuclear pores, which is dependent on DNA damage checkpoint kinases Mec1/Tel1 and replisome component Tof1. These findings suggest that rDNA-nuclear pore association is due to a replication fork block and subsequent DSB. Indeed, quantitative microscopy revealed that rDNA is relocated to the nuclear periphery upon induction of a DSB. Finally, rDNA stability was reduced in strains where this association with the nuclear envelope was prevented, which suggests its importance for avoiding improper recombination repair that could induce repeat instability.

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

The proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knock-out cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D.

The Nup84 complex coordinates the DNA damage response to warrant genome integrity.

DNA lesions interfere with cellular processes such as transcription and replication and need to be adequately resolved to warrant genome integrity. Beyond their primary role in molecule transport, nuclear pore complexes (NPCs) function in other processes such as transcription, nuclear organization and DNA double strand break (DSB) repair. Here we found that the removal of UV-induced DNA lesions by nucleotide excision repair (NER) is compromised in the absence of the Nup84 nuclear pore component. Importantly, nup84Δ cells show an exacerbated sensitivity to UV in early S phase and delayed replication fork progression, suggesting that unrepaired spontaneous DNA lesions persist during S phase. In addition, nup84Δ cells are defective in the repair of replication-born DSBs by sister chromatid recombination (SCR) and rely on post-replicative repair functions for normal proliferation, indicating dysfunctions in the cellular pathways that enable replication on damaged DNA templates. Altogether, our data reveal a central role of the NPC in the DNA damage response to facilitate replication progression through damaged DNA templates by promoting efficient NER and SCR and preventing chromosomal rearrangements.

Revised and Improved Procedure for Immunolocalization of Male Meiotic Chromosomal Proteins and Spindle in Plants without the Use of Enzymes.

Immunolocalization studies to visualize the distribution of proteins on meiotic chromosomes have become an integral part of studies on meiosis in the model organism Arabidopsis thaliana. These techniques have been used to visualize a wide range of meiotic proteins involved in different aspects of meiosis, including sister chromatid cohesion, recombination, synapsis, and chromosome segregation. However, the analysis of meiotic spindle structure by immunofluorescence is of outstanding importance in plant reproductive biology and is very challenging. In the following report, we describe the complete and easy protocol for the localization of proteins to the male meiotic spindle and male meiotic chromosomes. The protocol is fast, improved, and robust without the use of any harsh enzymes.

Tdp1 processes chromate-induced single-strand DNA breaks that collapse replication forks.

Hexavalent chromium [Cr(VI)] damages DNA and causes cancer, but it is unclear which DNA damage responses (DDRs) most critically protect cells from chromate toxicity. Here, genome-wide quantitative functional profiling, DDR measurements and genetic interaction assays in Schizosaccharomyces pombe reveal a chromate toxicogenomic profile that closely resembles the cancer chemotherapeutic drug camptothecin (CPT), which traps Topoisomerase 1 (Top1)-DNA covalent complex (Top1cc) at the 3’ end of single-stand breaks (SSBs), resulting in replication fork collapse. ATR/Rad3-dependent checkpoints that detect stalled and collapsed replication forks are crucial in Cr(VI)-treated cells, as is Mus81-dependent sister chromatid recombination (SCR) that repairs single-ended double-strand breaks (seDSBs) at broken replication forks. Surprisingly, chromate resistance does not require base excision repair (BER) or interstrand crosslink (ICL) repair, nor does co-elimination of XPA-dependent nucleotide excision repair (NER) and Rad18-mediated post-replication repair (PRR) confer chromate sensitivity in fission yeast. However, co-elimination of Tdp1 tyrosyl-DNA phosphodiesterase and Rad16-Swi10 (XPF-ERCC1) NER endonuclease synergistically enhances chromate toxicity in top1Δ cells. Pnk1 polynucleotide kinase phosphatase (PNKP), which restores 3’-hydroxyl ends to SSBs processed by Tdp1, is also critical for chromate resistance. Loss of Tdp1 ameliorates pnk1Δ chromate sensitivity while enhancing the requirement for Mus81. Thus, Tdp1 and PNKP, which prevent neurodegeneration in humans, repair an important class of Cr-induced SSBs that collapse replication forks.

Mre11 complex links sister chromatids to promote repair of a collapsed replication fork

Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11-Rad50-Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre112-Rad502 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.

SPARTAN promotes genetic diversification of the immunoglobulin-variable gene locus in avian DT40 cells

Prolonged replication arrest on damaged templates is a cause of fork collapse, potentially resulting in genome instability. Arrested replication is rescued by translesion DNA synthesis (TLS) and homologous recombination (HR)-mediated template switching. SPARTAN, a ubiquitin-PCNA-interacting regulator, regulates TLS via mechanisms incompletely understood. Here we show that SPARTAN promotes diversification of the chicken DT40 immunoglobulin-variable λ gene by facilitating TLS-mediated hypermutation and template switch-mediated gene-conversion, both induced by replication blocks at abasic sites. SPARTAN-/- and SPARTAN-/-/Polη-/-/Polζ-/- cells showed defective and similar decrease in hypermutation rates, as well as alterations in the mutation spectra, with decreased dG-to-dC transversions and increased dG-to-dA transitions. Strikingly, SPARTAN-/- cells also showed reduced template switch-mediated gene-conversion at the immunoglobulin locus, while being proficient in HR-mediated double strand break repair, and sister chromatid recombination. Notably, SPARTAN’s ubiquitin-binding zinc-finger 4 domain, but not the PCNA interacting peptide domain or its DNA-binding domain, was specifically required for the promotion of immunoglobulin gene-conversion, while all these three domains were shown to contribute similarly to TLS. In all, our results suggest that SPARTAN mediates TLS in concert with the Polη-Polζ pathway and that it facilitates HR-mediated template switching at a subset of stalled replication forks, potentially by interacting with unknown ubiquitinated proteins.

Alterations in Heterochromatic Knobs in Maize Callus Culture by Breakage-Fusion-Bridge Cycle and Unequal Crossing Over

The meiotic and mitotic behavior of regenerated plants derived from a long-term callus culture, designated 12-F, was analyzed. This culture was heterozygous for an amplification of the heterochromatic knob on the long arm of chromosome 7 (K7L). We aimed to investigate if the amplification resulted from a breakage-fusion-bridge (BFB) cycle or from unequal sister chromatid recombination. Therefore, C-banded mitotic metaphases and pachytene, diakinesis, and anaphase I of regenerated plants were analyzed. Additionally, the occurrence of alterations in K7L was investigated in C-banded metaphases from short-term callus cultures derived from lines related to the donor genotype of the 12-F culture. As a result, plants homozygous and heterozygous for the amplification were detected. Meiosis was normal with few abnormalities, such as a low frequency of univalents at diakinesis. In the callus cultures a chromosome 7 with knobs of different sizes in the sister chromatids was detected and interpreted as a result of unequal crossing over. Other chromosomal alterations were consistent with the occurrence of BFB cycles. The finding of unequal crossing over in the cultures supports the conclusion that the amplification in the culture 12-F would be derived from this mechanism. If the amplification was derived from a BFB cycle, the terminal euchromatic segment between knob and the telomere would be deleted, and possibly, homozygous plants would not be viable.

Genome-wide mapping of sister chromatid exchange events in single yeast cells using Strand-seq.

Homologous recombination involving sister chromatids is the most accurate, and thus most frequently used, form of recombination-mediated DNA repair. Despite its importance, sister chromatid recombination is not easily studied because it does not result in a change in DNA sequence, making recombination between sister chromatids difficult to detect. We have previously developed a novel DNA template strand sequencing technique, called Strand-seq, that can be used to map sister chromatid exchange (SCE) events genome-wide in single cells. An increase in the rate of SCE is an indicator of elevated recombination activity and of genome instability, which is a hallmark of cancer. In this study, we have adapted Strand-seq to detect SCE in the yeast Saccharomyces cerevisiae. We provide the first quantifiable evidence that most spontaneous SCE events in wild-type cells are not due to the repair of DNA double-strand breaks.

Lingering single-strand breaks trigger Rad51-independent homology-directed repair of collapsed replication forks in the polynucleotide kinase/phosphatase mutant of fission yeast.

The DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) protects genome integrity by restoring ligatable 5’-phosphate and 3’-hydroxyl termini at single-strand breaks (SSBs). In humans, PNKP mutations underlie the neurological disease known as MCSZ, but these individuals are not predisposed for cancer, implying effective alternative repair pathways in dividing cells. Homology-directed repair (HDR) of collapsed replication forks was proposed to repair SSBs in PNKP-deficient cells, but the critical HDR protein Rad51 is not required in PNKP-null (pnk1Δ) cells of Schizosaccharomyces pombe. Here, we report that pnk1Δ cells have enhanced requirements for Rad3 (ATR/Mec1) and Chk1 checkpoint kinases, and the multi-BRCT domain protein Brc1 that binds phospho-histone H2A (γH2A) at damaged replication forks. The viability of pnk1Δ cells depends on Mre11 and Ctp1 (CtIP/Sae2) double-strand break (DSB) resection proteins, Rad52 DNA strand annealing protein, Mus81-Eme1 Holliday junction resolvase, and Rqh1 (BLM/WRN/Sgs1) DNA helicase. Coupled with increased sister chromatid recombination and Rad52 repair foci in pnk1Δ cells, these findings indicate that lingering SSBs in pnk1Δ cells trigger Rad51-independent homology-directed repair of collapsed replication forks. From these data, we propose models for HDR-mediated tolerance of persistent SSBs with 3’ phosphate in pnk1Δ cells.

FANCJ helicase controls the balance between short- and long-tract gene conversions between sister chromatids.

The FANCJ DNA helicase is linked to hereditary breast and ovarian cancers as well as bone marrow failure disorder Fanconi anemia (FA). Although FANCJ has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), the molecular mechanism underlying the tumor suppressor functions of FANCJ remains obscure. Here, we demonstrate that FANCJ deficient human and hamster cells exhibit reduction in the overall gene conversions in response to a site-specific chromosomal DSB induced by I-SceI endonuclease. Strikingly, the gene conversion events were biased in favour of long-tract gene conversions in FANCJ depleted cells. The fine regulation of short- (STGC) and long-tract gene conversions (LTGC) by FANCJ was dependent on its interaction with BRCA1 tumor suppressor. Notably, helicase activity of FANCJ was essential for controlling the overall HR and in terminating the extended repair synthesis during sister chromatid recombination (SCR). Moreover, cells expressing FANCJ pathological mutants exhibited defective SCR with an increased frequency of LTGC. These data unravel the novel function of FANCJ helicase in regulating SCR and SCR associated gene amplification/duplications and imply that these functions of FANCJ are crucial for the genome maintenance and tumor suppression.

GSK-3β Homolog Rim11 and the Histone Deacetylase Complex Ume6-Sin3-Rpd3 Are Involved in Replication Stress Response Caused by Defects in Dna2.

Lagging strand synthesis is mechanistically far more complicated than leading strand synthesis because it involves multistep processes and requires considerably more enzymes and protein factors. Due to this complexity, multiple fail-safe factors are required to ensure successful replication of the lagging strand DNA. We attempted to identify novel factors that are required in the absence of the helicase activity of Dna2, an essential enzyme in Okazaki-fragment maturation. In this article, we identified Rim11, a GSK-3β-kinase homolog, as a multicopy suppressor of dna2 helicase-dead mutant (dna2-K1080E). Subsequent epistasis analysis revealed that Ume6 (a DNA binding protein, a downstream substrate of Rim11) also acted as a multicopy suppressor of the dna2 allele. We found that the interaction of Ume6 with the conserved histone deacetylase complex Sin3-Rpd3 and the catalytic activity of Rpd3 were indispensable for the observed suppression of the dna2 mutant. Moreover, multicopy suppression by Rim11/Ume6 requires the presence of sister-chromatid recombination mediated by Rad52/Rad59 proteins, but not vice versa. Interestingly, the overexpression of Rim11 or Ume6 also suppressed the MMS sensitivity of rad59Δ. We also showed that the lethality of dna2 helicase-dead mutant was attributed to checkpoint activation and that decreased levels of deoxynucleotide triphosphates (dNTPs) by overexpressing Sml1 (an inhibitor of ribonucleotide reductase) rescued the dna2 mutant. We also present evidence that indicates Rim11/Ume6 works independently but in parallel with that of checkpoint inhibition, dNTP regulation, and sister-chromatid recombination. In conclusion, our results establish Rim11, Ume6, the histone deacetylase complex Sin3-Rpd3 and Sml1 as new factors important in the events of faulty lagging strand synthesis.