Unraveling MLL1-fusion Leukemia: Epigenetic Revelations from an iPS Cell Point Mutation.

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 mono- and dimethylation), remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition (EMT) phenotypes of MLL1F-LSCs- without requiring a translocation or functional wild-type MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long non-coding RNA (lncRNA). EMT markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1's enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Complex interplay between FMRP and DHX9 during DNA replication stress.

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.

Deregulation of Ribosome Biogenesis in Nitrite-Oxidizing Bacteria Leads to Nitrite Accumulation.

Nitrite (NO2-) accumulation caused by nitrite-oxidizing bacteria (NOB) inhibition in nitrification is a double-edged sword, i.e., a disaster in aquatic environments but a hope for innovating nitrogen removal technology in wastewater treatment. However, little information is available regarding the molecular mechanism of NOB inhibition at the cellular level. Herein, we investigate the response of NOB inhibition on NO2- accumulation established by a side-stream free ammonia treatment unit in a nitrifying reactor using integrated metagenomics and metaproteomics. Results showed that compared with the baseline, the relative abundance and activity of NOB in the experimental stage decreased by 91.64 and 68.66%, respectively, directly resulting in a NO2- accumulation rate of 88%. Moreover, RNA polymerase, translation factors, and aa-tRNA ligase were significantly downregulated, indicating that protein synthesis in NOB was interfered during NO2- accumulation. Further investigations showed that ribosomal proteins and GTPases, responsible for bindings between either ribosomal proteins and rRNA or ribosome subunits, were remarkably downregulated. This suggests that ribosome biogenesis was severely disrupted, which might be the key reason for the inhibited protein synthesis. Our findings fill a knowledge gap regarding the underlying mechanisms of NO2- accumulation, which would be beneficial for regulating the accumulation of NO2- in aquatic environments and engineered systems.

Rtt107 Is a Multi-functional Scaffold Supporting Replication Progression with Partner SUMO and Ubiquitin Ligases.

Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.

The yeast Dbf4 Zn2+ finger domain suppresses single-stranded DNA at replication forks initiated from a subset of origins.

Dbf4 is the cyclin-like subunit for the Dbf4-dependent protein kinase (DDK), required for activating the replicative helicase at DNA replication origin that fire during S phase. Dbf4 also functions as an adaptor, targeting the DDK to different groups of origins and substrates. Here we report a genome-wide analysis of origin firing in a budding yeast mutant, dbf4-zn, lacking the Zn2+ finger domain within the C-terminus of Dbf4. At one group of origins, which we call dromedaries, we observe an unanticipated DNA replication phenotype: accumulation of single-stranded DNA spanning ± 5kbp from the center of the origins. A similar accumulation of single-stranded DNA at origins occurs more globally in pri1-m4 mutants defective for the catalytic subunit of DNA primase and rad53 mutants defective for the S phase checkpoint following DNA replication stress. We propose the Dbf4 Zn2+ finger suppresses single-stranded gaps at replication forks emanating from dromedary origins. Certain origins may impose an elevated requirement for the DDK to fully initiate DNA synthesis following origin activation. Alternatively, dbf4-zn may be defective for stabilizing/restarting replication forks emanating from dromedary origins during replication stress.

Yeast Stn1 promotes MCM to circumvent Rad53 control of the S phase checkpoint.

Treating yeast cells with the replication inhibitor hydroxyurea activates the S phase checkpoint kinase Rad53, eliciting responses that block DNA replication origin firing, stabilize replication forks, and prevent premature extension of the mitotic spindle. We previously found overproduction of Stn1, a subunit of the telomere-binding Cdc13-Stn1-Ten1 complex, circumvents Rad53 checkpoint functions in hydroxyurea, inducing late origin firing and premature spindle extension even though Rad53 is activated normally. Here, we show Stn1 overproduction acts through remarkably similar pathways compared to loss of RAD53, converging on the MCM complex that initiates origin firing and forms the catalytic core of the replicative DNA helicase. First, mutations affecting Mcm2 and Mcm5 block the ability of Stn1 overproduction to disrupt the S phase checkpoint. Second, loss of function stn1 mutations compensate rad53 S phase checkpoint defects. Third Stn1 overproduction suppresses a mutation in Mcm7. Fourth, stn1 mutants accumulate single-stranded DNA at non-telomeric genome locations, imposing a requirement for post-replication DNA repair. We discuss these interactions in terms of a model in which Stn1 acts as an accessory replication factor that facilitates MCM activation at ORIs and potentially also maintains MCM activity at replication forks advancing through challenging templates.

Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.

We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

Incision of damaged DNA in the presence of an impaired Smc5/6 complex imperils genome stability.

The Smc5/6 complex is implicated in homologous recombination-mediated DNA repair during DNA damage or replication stress. Here, we analysed genome-wide replication dynamics in a hypomorphic budding yeast mutant, smc6-P4 The overall replication dynamics in the smc6 mutant is similar to that in the wild-type cells. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We tested if inhibiting the ribonucleotide excision repair pathway would exacerbate the smc6 mutant in response to DNA replication stress. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant's hypersensitivity to induced replication stress. We propose that nucleotide incision in the absence of a functional Smc5/6 complex has more disastrous outcomes than the damage per se Our study provides novel perspectives for the role of the Smc5/6 complex during DNA replication.

Fragility Extraordinaire: Unsolved Mysteries of Chromosome Fragile Sites.

Chromosome fragile sites are a fascinating cytogenetic phenomenon now widely implicated in a slew of human diseases ranging from neurological disorders to cancer. Yet, the paths leading to these revelations were far from direct, and the number of fragile sites that have been molecularly cloned with known disease-associated genes remains modest. Moreover, as more fragile sites were being discovered, research interests in some of the earliest discovered fragile sites ebbed away, leaving a number of unsolved mysteries in chromosome biology. In this review we attempt to recount some of the early discoveries of fragile sites and highlight those phenomena that have eluded intense scrutiny but remain extremely relevant in our understanding of the mechanisms of chromosome fragility. We then survey the literature for disease association for a comprehensive list of fragile sites. We also review recent studies addressing the underlying cause of chromosome fragility while highlighting some ongoing debates. We report an observed enrichment for R-loop forming sequences in fragile site-associated genes than genomic average. Finally, we will leave the reader with some lingering questions to provoke discussion and inspire further scientific inquiries.

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

A Double Jeopardy: Loss of FMRP Results in DSB and Down-regulated DNA Repair.

Our understanding of the molecular functions of the nucleocytoplasmic FMRP protein, which, if absent or dysfunctional, causes the fragile X syndrome (FXS), largely revolves around its involvement in protein translation regulation in the cytoplasm. Recent studies have begun honing in on the nuclear and genomic functions of FMRP. We have shown that during DNA replication stress, cells derived from FXS patients sustain increased level of R-loop formation and DNA double strand breaks. Here, we describe a transcriptomic analysis of these cells in order to identify those genes most impacted by the loss of FMRP with and without replication stress. We show that FMRP loss causes transcriptomic changes previously reported in untreated conditions. Importantly, we also show that replication stress, in addition to causing excess of DSB, results in down-regulation of transcription in virtually all DNA repair pathways. This finding suggests that despite normal DNA damage response, FXS patient-derived cells experience R-loop-induced DNA breakage as well as impaired DNA repair functions, effectively a double jeopardy. We suggest that it is imperative to deepen the understanding of the nuclear functions, particularly a genome protective function, of FMRP, which will lead to discoveries of novel therapeutic interventions for the FXS.

Identification of Recurrent Chromosome Breaks Underlying Structural Rearrangements in Mammary Cancer Cell Lines.

Cancer genomes are characterized by the accumulation of small-scale somatic mutations as well as large-scale chromosomal deletions, amplifications, and complex structural rearrangements. This characteristic is at least partially dependent on the ability of cancer cells to undergo recurrent chromosome breakage. In order to address the extent to which chromosomal structural rearrangement breakpoints correlate with recurrent DNA double-strand breaks (DSBs), we simultaneously mapped chromosome structural variation breakpoints (using whole-genome DNA-seq) and spontaneous DSB formation (using Break-seq) in the estrogen receptor (ER)-positive breast cancer cell line MCF-7 and a non-cancer control breast epithelium cell line MCF-10A. We identified concurrent DSBs and structural variation breakpoints almost exclusively in the pericentromeric region of chromosome 16q in MCF-7 cells. We fine-tuned the identification of copy number variation breakpoints on 16q. In addition, we detected recurrent DSBs that occurred in both MCF-7 and MCF-10A. We propose a model for DSB-driven chromosome rearrangements that lead to the translocation of 16q, likely with 10q, and the eventual 16q loss that does not involve the pericentromere of 16q. We present evidence from RNA-seq data that select genes, including SHCBP1, ORC6, and MYLK3, which are immediately downstream from the 16q pericentromere, show heightened expression in MCF-7 cell line compared to the control. Data published by The Cancer Genome Atlas show that all three genes have increased expression in breast tumor samples. We found that SHCBP1 and ORC6 are both strong poor prognosis and treatment outcome markers in the ER-positive breast cancer cohort. We suggest that these genes are potential oncogenes for breast cancer progression. The search for tumor suppressor loss that accompanies the 16q loss ought to be augmented by the identification of potential oncogenes that gained expression during chromosomal rearrangements.

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains.

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two yeast strains challenged by hydroxyurea, an inhibitor of the ribonucleotide reductase. We gained understanding of the impact on origin activation by three factors: S-phase checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Wild type W303 showed a significant reduction of fork progression accompanied by an elevated level of Rad53 phosphorylation as well as physical presence at origins compared to A364a. Moreover, a rad53K227A mutant in W303 activated more origins, accompanied by global reduction of ssDNA across all origins, compared to A364a. Sequence polymorphism in the consensus motifs of origins plays a minor role in determining strain-specific activity. Finally, we identified a new class of origins only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". Our study presents a comprehensive list of differentially used origins and provide new insights into the mechanisms of origin activation.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks.

Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300 kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

Genome-wide mapping of DNA double-strand breaks from eukaryotic cell cultures using Break-seq.

We describe a genome-wide DNA double-strand break (DSB) mapping technique, Break-seq. In this protocol, we provide step-by-step instructions for cell embedment in agarose, in-gel DSB labeling and subsequent capture, followed by standard Illumina library construction and sequencing. We also provide the framework for sequence data processing and DSB peak identification. Finally, we present a custom-designed 3D-printed device for processing agarose-embedded DNA samples. The protocol is applicable to Saccharomyces cerevisiae, as well as mammalian suspension, adherent, and 3D organoid cell cultures. For complete details on the use and execution of this protocol, please refer to Hoffman et al. (2015) and Chakraborty et al. (2020).

R-loops at centromeric chromatin contribute to defects in kinetochore integrity and chromosomal instability in budding yeast.

R-loops, the byproduct of DNA-RNA hybridization and the displaced single-stranded DNA (ssDNA), have been identified in bacteria, yeasts, and other eukaryotic organisms. The persistent presence of R-loops contributes to defects in DNA replication and repair, gene expression, and genomic integrity. R-loops have not been detected at centromeric (CEN) chromatin in wild-type budding yeast. Here we used an hpr1∆ strain that accumulates R-loops to investigate the consequences of R-loops at CEN chromatin and chromosome segregation. We show that Hpr1 interacts with the CEN-histone H3 variant, Cse4, and prevents the accumulation of R-loops at CEN chromatin for chromosomal stability. DNA-RNA immunoprecipitation (DRIP) analysis showed an accumulation of R-loops at CEN chromatin that was reduced by overexpression of RNH1 in hpr1∆ strains. Increased levels of ssDNA, reduced levels of Cse4 and its assembly factor Scm3, and mislocalization of histone H3 at CEN chromatin were observed in hpr1∆ strains. We determined that accumulation of R-loops at CEN chromatin contributes to defects in kinetochore biorientation and chromosomal instability (CIN) and these phenotypes are suppressed by RNH1 overexpression in hpr1∆ strains. In summary, our studies provide mechanistic insights into how accumulation of R-loops at CEN contributes to defects in kinetochore integrity and CIN.

A Tale of Loops and Tails: The Role of Intrinsically Disordered Protein Regions in R-Loop Recognition and Phase Separation.

R-loops are non-canonical, three-stranded nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under both normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, with many of these proteins containing folded nucleic acid processing and binding domains that prevent (e.g., topoisomerases), resolve (e.g., helicases, nucleases), or recognize (e.g., KH, RRMs) R-loops. However, a significant number of these R-loop interacting Enzyme and Reader proteins also contain long stretches of intrinsically disordered regions (IDRs). The precise molecular and structural mechanisms by which the folded domains and IDRs synergize to recognize and process R-loops or modulate R-loop-mediated signaling have not been fully explored. While studying one such modular R-loop Reader, the Fragile X Protein (FMRP), we unexpectedly discovered that the C-terminal IDR (C-IDR) of FMRP is the predominant R-loop binding site, with the three N-terminal KH domains recognizing the trailing ssRNA overhang. Interestingly, the C-IDR of FMRP has recently been shown to undergo spontaneous Liquid-Liquid Phase Separation (LLPS) assembly by itself or in complex with another non-canonical nucleic acid structure, RNA G-quadruplex. Furthermore, we have recently shown that FMRP can suppress persistent R-loops that form during transcription, a process that is also enhanced by LLPS via the assembly of membraneless transcription factories. These exciting findings prompted us to explore the role of IDRs in R-loop processing and signaling proteins through a comprehensive bioinformatics and computational biology study. Here, we evaluated IDR prevalence, sequence composition and LLPS propensity for the known R-loop interactome. We observed that, like FMRP, the majority of the R-loop interactome, especially Readers, contains long IDRs that are highly enriched in low complexity sequences with biased amino acid composition, suggesting that these IDRs could directly interact with R-loops, rather than being "mere flexible linkers" connecting the "functional folded enzyme or binding domains". Furthermore, our analysis shows that several proteins in the R-loop interactome are either predicted to or have been experimentally demonstrated to undergo LLPS or are known to be associated with phase separated membraneless organelles. Thus, our overall results present a thought-provoking hypothesis that IDRs in the R-loop interactome can provide a functional link between R-loop recognition via direct binding and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci. The absence or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the widespread R-loop-mediated DNA double strand breaks that we recently observed in Fragile X patient-derived cells.

Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

Inhibition of spindle extension through the yeast S phase checkpoint is coupled to replication fork stability and the integrity of centromeric DNA.

Budding yeast treated with hydroxyurea (HU) activate the S phase checkpoint kinase Rad53, which prevents DNA replication forks from undergoing aberrant structural transitions and nuclease processing. Rad53 is also required to prevent premature extension of the mitotic spindle that assembles during a HU-extended S phase. Here we present evidence that checkpoint restraint of spindle extension is directly coupled to Rad53 control of replication fork stability. In budding yeast, centromeres are flanked by replication origins that fire in early S phase. Mutations affecting the Zn2+-finger of Dbf4, an origin activator, preferentially reduce centromere-proximal origin firing in HU, corresponding with suppression of rad53 spindle extension. Inactivating Exo1 nuclease or displacing centromeres from origins provides a similar suppression. Conversely, short-circuiting Rad53 targeting of Dbf4, Sld3, and Dun1, substrates contributing to fork stability, induces spindle extension. These results reveal spindle extension in HU-treated rad53 mutants is a consequence of replication fork catastrophes at centromeres. When such catastrophes occur, centromeres become susceptible to nucleases, disrupting kinetochore function and spindle force balancing mechanisms. At the same time, our data indicate centromere duplication is not required to stabilize S phase spindle structure, leading us to propose a model for how monopolar kinetochore-spindle attachments may contribute to spindle force balance in HU.

Schizosacchromyces pombe Dpb2 binds to origin DNA early in S phase and is required for chromosomal DNA replication.

Genetic evidence suggests that DNA polymerase epsilon (Pol epsilon) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol epsilon in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol epsilon associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol epsilon is not dependent on its DNA synthesis activity.