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The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics.

Replication timing organizes epigenome The temporal order of DNA replication is conserved from yeast to humans, but its biological significance remains unclear. Klein et al. eliminated the protein RIF1, a master regulator of replication timing, in several human cell lines. RIF1 loss during the G1 phase of the cell cycle resulted in a heterogeneous, nearly random replication timing program from the first S phase that persisted even in stable RIF1-null clones. Altered replication timing was followed by replication-dependent redistribution of active and repressive histone modifications and alterations in genome architecture. These results support a model in which replication timing orchestrates the epigenetic state of newly replicated chromatin. Science , this issue p. 371

To ensure faultless duplication of the entire genome, eukaryotic replication initiates from thousands of replication origins. Replication forks emanating from origins move through the chromatin until they encounter forks from neighbouring origins, at which point they terminate. In the final stages of this process the replication machinery (replisome) is unloaded from chromatin and disassembled. Work from model organisms has elucidated that during replisome unloading, the MCM7 subunit of the terminated replicative helicase is polyubiquitylated and processed by p97/VCP segregase, leading to disassembly of the helicase and the replisome, which is built around it. In higher eukaryotes (worms, frogs, mouse embryonic stem cells), MCM7 ubiquitylation is driven by a Cullin2-based ubiquitin ligase, with LRR1 as a substrate receptor. To date, most of our knowledge of replication termination comes from model organisms and embryonic systems and little is known about how this process is executed and regulated in human somatic cells. Here we thus established methods to study replisome disassembly in human model cell lines. Using flow cytometry, immunofluorescence microscopy and chromatin isolation with western blotting, we can visualise unloading of the replisome (MCM7 and CDC45) from chromatin by the end of S-phase. We observe interaction of replicative helicase (CMG complex) with CUL2LRR1 and ubiquitylation of MCM7 on chromatin, specifically in S-phase, suggesting that this is a replication-dependent modification. Importantly, we are able to show that replisome disassembly in this system also requires Cullin2, LRR1 and p97, demonstrating conservation of the mechanism. Moreover, we present evidence that the back-up mitotic replisome disassembly pathway is also recapitulated in human somatic cells. Finally, while we find that treatment with small molecule inhibitors against cullin-based ubiquitin ligases (CULi) and p97 (p97i) does lead to phenotypes of replisome disassembly defects, they also both lead to induction of replication stress in somatic cells, which limits their usefulness as tools to specifically target replisome disassembly processes in this setting.

Cancer stem‐like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c‐Myc expression induces stronger DNA replication stress in patient‐derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini‐chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c‐Myc. c‐Myc‐dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient‐derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S‐phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c‐Myc‐MCM10 axis for preventing drug resistance and recurrence of tumors., We provide evidence that upregulated c‐Myc expression induces stronger DNA replication stress in patient‐derived breast cancer stem‐like cells than in differentiated cancer cells. Our results suggest critical roles for mini‐chromosome maintenance protein 10 (MCM10), which is a firing (activating) factor of the DNA replication origins, to compensate for the DNA replication stress.

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, for the first time, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We find that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequence (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on both co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands in both co- directional and convergent forks is compromised by transcription. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability.

Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15–45 min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR–Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.

MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.

Iemura et al. find that chromosome oscillation during metaphase facilitates correction of erroneous kinetochore–microtubule attachments through Hec1 phosphorylation by Aurora A on the spindle. Chromosome oscillation is attenuated in cancer cell lines, which may be a cause of chromosomal instability., Most cancer cells show chromosomal instability, a condition where chromosome missegregation occurs frequently. We found that chromosome oscillation, an iterative chromosome motion during metaphase, is attenuated in cancer cell lines. We also found that metaphase phosphorylation of Hec1 at serine 55, which is mainly dependent on Aurora A on the spindle, is reduced in cancer cell lines. The Aurora A–dependent Hec1-S55 phosphorylation level was regulated by the chromosome oscillation amplitude and vice versa: Hec1-S55 and -S69 phosphorylation by Aurora A is required for efficient chromosome oscillation. Furthermore, enhancement of chromosome oscillation reduced the number of erroneous kinetochore–microtubule attachments and chromosome missegregation, whereas inhibition of Aurora A during metaphase increased such errors. We propose that Aurora A–mediated metaphase Hec1-S55 phosphorylation through chromosome oscillation, together with Hec1-S69 phosphorylation, ensures mitotic fidelity by eliminating erroneous kinetochore–microtubule attachments. Attenuated chromosome oscillation and the resulting reduced Hec1-S55 phosphorylation may be a cause of CIN in cancer cell lines., Graphical Abstract

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here, we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.

Cancer stem-like cells (CSCs) are responsible for the drug resistance of tumors and recurrence while they experience DNA replication stress. However, the underlying mechanisms that cause DNA replication stress in CSCs and how they compensate for this stress remain unclear. Here we provide evidence that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), which is a firing (activating) factor of the DNA replication origins, to compensate for the DNA replication stress. Expression levels of MCM10 are upregulated in CSCs and maintained by c-Myc. c-Myc-dependent collisions may take place between RNA transcription and DNA replication machinery in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to the collisions to ensure replication progression. Moreover, patient-derived breast CSCs were dependent on MCM10 for their maintenance even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. In addition, MCM10 depletion decreased the growth of cancer cells but not normal cells. Therefore, MCM10 is likely to robustly compensate for DNA replication stress and facilitate genome duplication in the S-phase in cancer cells, which is more pronounced in CSCs. We provide a preclinical rationale to target the c-Myc-MCM10 axis to prevent drug resistance and recurrence.

As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.

The conditional depletion of a protein of interest (POI) is useful not only for loss-of-function studies, but also for the modulation of biological pathways. Technologies that work at the level of DNA, mRNA, and protein are available for temporal protein depletion. Compared with technologies targeting the pretranslation steps, direct protein depletion (or protein knockdown approaches) is advantageous in terms of specificity, reversibility, and time required for depletion, which can be achieved by fusing a POI with a protein domain called a degron that induces rapid proteolysis of the fusion protein. Conditional degrons can be activated or inhibited by temperature, small molecules, light, or the expression of another protein. The conditional degron-based technologies currently available are described and discussed.

DNA is replicated in a defined temporal order termed the replication timing (RT) program. RT is spatially segregated in the nucleus with early/late replication corresponding to Hi-C A/B chromatin compartments, respectively. Early replication is also associated with active histone modifications and transcriptional permissiveness. However, the mechanistic interplay between RT, chromatin state, and genome compartmentalization is largely unknown. Here we report that RT is central to epigenome maintenance and compartmentalization in both human embryonic stem cells (hESCs) and cancer cell line HCT116. Knockout (KO) of the conserved RT control factor RIF1, rather than causing discrete RT switches as previously suspected, lead to dramatically increased cell to cell heterogeneity of RT genome wide, despite RIF1’s enrichment in late replicating chromatin. RIF1 KO hESCs have a nearly random RT program, unlike all prior RIF1 KO cells, including HCT116, which show localized alterations. Regions that retain RT, which are prevalent in HCT116 but rare in hESCs, consist of large H3K9me3 domains revealing two independent mechanisms of RT regulation that are used to different extents in different cell types. RIF1 KO results in a striking genome wide downregulation of H3K27ac peaks and enrichment of H3K9me3 at large domains that remain late replicating, while H3K27me3 and H3K4me3 are re-distributed genome wide in a cell type specific manner. These histone modification changes coincided with global reorganization of genome compartments, transcription changes and a genome wide strengthening of TAD structures. Inducible degradation of RIF1 revealed that disruption of RT is upstream of genome compartmentalization changes. Our findings demonstrate that disruption of RT leads to widespread epigenetic mis-regulation, supporting previously speculative models in which the timing of chromatin assembly at the replication fork plays a key role in maintaining the global epigenetic state, which in turn drives genome architecture.

SUMMARY Human RIF1 functions in DNA replication and damage repair. Of clinical interest, RIF1-depleted cells are highly sensitive to replication inhibitors such as Aphidicolin, but the reasons for this sensitivity have been enigmatic. Here we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. RIF1 Long and Short isoforms are produced by alternative splicing. We find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is completely incapable of mediating protection. Consistent with its isoform-specific role in enabling survival, RIF1-Long is specifically required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages to support genome maintenance following replication insult, with the RIF1-Long isoform playing a previously unsuspected but crucial role in damage site protection during the ensuing G1 phase.

SummaryStudying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function.

At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.

Chk1 is an evolutionally conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell-cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) using a CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell-cycle arrest at S or G2 phase. Cdc25A stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell-cycle progression mainly by inducing Cdc25A degradation.

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA’s N-terminal long arm, dynein-based astral microtubule gliding, and NuMA’s direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment/activation by NuMA’s N-terminal long arm, and NuMA’s direct microtubule-binding activities to achieve a multiplicity of microtubule interactions. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.

Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.

Single-cell analyses in budding yeast reveal that neighboring replicons are assembled stochastically and stay associated to maintain stable replication factories., Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

Summary Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3–Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2–Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites., Graphical Abstract, Highlights • The Ctf19 complex recruits Cdc7-Dbf4 kinase (DDK) to kinetochores (KTs) in telophase • DDK at KTs recruits Sld3–Sld7 to pericentromeric origins, advancing replication timing • DDK at KTs recruits Scc2, enhancing cohesin loading and cohesion at pericentromeres • The DDK functions in replication timing and cohesion are independent of each other

Centromeres are known to promote kinetochore assembly. However, two other roles—facilitating robust sister chromatid cohesion and advancing the replication timing of centromeric regions—have recently emerged. The three are analysed in this Review., The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister-chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore–microtubule interaction, focusing on sister-kinetochore bi-orientation (or chromosome bi-orientation). We also discuss the biological importance of robust pericentromeric cohesion and early centromere replication, as well as the mechanisms orchestrating these two functions at the microtubule attachment site.

Schizosaccharomyces pombe rad2 is involved in Okazaki fragments processing during lagging-strand DNA replication. Previous studies identified several slr mutants that are co-lethal with rad2Delta and sensitive to methyl methanesulfonate as single mutants. One of these mutants, slr3-1, is characterized here. Complementation and sequence analyses show that slr3-1 (mcl1-101) is allelic to mcl1(+), which is required for chromosome replication, cohesion and segregation. mcl1-101 is temperature-sensitive for growth and is highly sensitive to DNA damage. mcl1 cells arrest with 2C DNA content and chromosomal DNA double-strand breaks accumulate at the restrictive temperature. Mcl1p, which belongs to the Ctf4p/SepBp family, interacts both genetically and physically with DNA polymerase alpha. Mutations in rhp51 and dna2 enhance the growth defect of the mcl1-101 mutant. These results strongly suggest that Mcl1p is a functional homologue of Saccharomyces cerevisiae Ctf4p and plays a role in lagging-strand synthesis and Okazaki fragment processing, in addition to DNA repair.

Gene targeting to knock out the activity of specific genes has become important due to recent progress in genomics research. But this technique is still unavailable for many organisms, including economically important microorganisms, due to the high background of ectopic integration during genetic transformation. Strategies to improve targeting efficiency have included manipulating the expression of genes that are involved in homologous recombination. In this study, transcription of Aspergillus nidulans uvsC was elevated using the promoter sequences of the glyceraldehyde-3-phosphate dehydrogenase and Taka-amylase A genes from A. nidulans and A. oryzea respectively. Although a several-fold increase in the efficiency of targeting was observed at 3 loci, mycelial growth was suppressed in strains that had higher levels of uvsC transcription. These results suggest that uvsC is a rate-limiting factor in gene targeting, and that the increased efficiency of this targeting is hindered by a negative effect of increased transcription on cell proliferation.