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Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome-wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild-type Rif1 and truncated Rif1 lacking the Rap1-interaction domain, we identify hundreds of Rap1-dependent and Rap1-independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1-independent manner, associating with both early and late-initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

During DNA replication, thousands of replication origins are activated across the genome. Chromatin architecture contributes to origin specification and usage, yet it remains unclear which chromatin features impact on DNA replication. Here, we perform a RNAi screen for chromatin regulators implicated in replication control by measuring RPA accumulation upon replication stress. We identify six factors required for normal rates of DNA replication and characterize a function of the bromodomain and PHD finger-containing protein 3 (BRPF3) in replication initiation. BRPF3 forms a complex with HBO1 that specifically acetylates histone H3K14, and genomewide analysis shows high enrichment of BRPF3, HBO1 and H3K14ac at ORC1-binding sites and replication origins found in the vicinity of TSSs. Consistent with this, BRPF3 is necessary for H3K14ac at selected origins and efficient origin activation. CDC45 recruitment, but not MCM2-7 loading, is impaired in BRPF3-depleted cells, identifying a BRPF3-dependent function of HBO1 in origin activation that is complementary to its role in licencing. We thus propose that BRPF3-HBO1 acetylation of histone H3K14 around TSS facilitates efficient activation of nearby replication origins., (© 2015 The Authors.)

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of "firing factors." Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins.

Eukaryotic chromosomes are replicated from multiple origins that initiate throughout the S-phase of the cell cycle. Why all origins do not fire simultaneously at the beginning of S-phase is not known, but two kinase activities, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), are continually required throughout the S-phase for all replication initiation events. Here, we show that the two CDK substrates Sld3 and Sld2 and their binding partner Dpb11, together with the DDK subunit Dbf4 are in low abundance in the budding yeast, Saccharomyces cerevisiae. Over-expression of these factors is sufficient to allow late firing origins of replication to initiate early and together with deletion of the histone deacetylase RPD3, promotes the firing of heterochromatic, dormant origins. We demonstrate that the normal programme of origin firing prevents inappropriate checkpoint activation and controls S-phase length in budding yeast. These results explain how the competition for limiting DDK kinase and CDK targets at origins regulates replication initiation kinetics during S-phase and establishes a unique system with which to investigate the biological roles of the temporal programme of origin firing.

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci-spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid.

The checkpoint kinase 1 (Chk1) preserves genome integrity when replication is performed on damaged templates. Recently, Chk1 has also been implicated in regulating different aspects of unperturbed S phase. Using mammalian and avian cells with compromised Chk1 activity, we show that an increase in active replicons compensates for inefficient DNA polymerisation. In the absence of damage, loss of Chk1 activity correlates with the frequent stalling and, possibly, collapse of active forks and activation of adjacent, previously suppressed, origins. In human cells, super-activation of replication origins is restricted to pre-existing replication factories. In avian cells, in contrast, Chk1 deletion also correlates with the super-activation of replication factories and loss of temporal continuity in the replication programme. The same phenotype is induced in wild-type avian cells when Chk1 or ATM/ATR is inhibited. These observations show that Chk1 regulates replication origin activation and contributes to S-phase progression in somatic vertebrate cells.

The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Homologous recombination safeguards genome integrity, but it can also cause genome instability of important consequences for cell proliferation and organism development. Transcription induces recombination, as shown in prokaryotes and eukaryotes for both spontaneous and developmentally regulated events such as those responsible for immunoglobulin class switching. Deciphering the molecular basis of transcription-associated recombination (TAR) is important in understanding genome instability. Using novel plasmid-borne recombination constructs in Saccharomyces cerevisiae, we show that RNA polymerase II (RNAPII) transcription induces recombination by impairing replication fork progression. RNAPII transcription concomitant to head-on oncoming replication causes a replication fork pause (RFP) that is linked to a significant increase in recombination. However, transcription that is codirectional with replication has little effect on replication fork progression and recombination. Transcription occurring in the absence of replication does not affect either recombination or replication fork progression. The Rrm3 helicase, which is required for replication fork progression through nucleoprotein complexes, facilitates replication through the transcription-dependent RFP site and reduces recombination. Therefore, our work provides evidence that one mechanism responsible for TAR is RNAP-mediated replication impairment.

During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization.

The presence of multiple clusters of runs of asymmetric adenine or thymine is a feature commonly found in eukaryotic replication origins. Here we report that the helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine stretches. The Mcm helicase is specifically activated by a synthetic bubble structure which mimics an activated replication origin, as well as by a Y-fork structure, provided that a single-stranded DNA region of sufficient length is present in the unwound segment or 3' tail, respectively, and that it carries clusters of thymines. Sequences derived from the human lamin B2 origin can serve as a potent activator for the Mcm helicase, and substitution of its thymine clusters with guanine leads to loss of this activation. At the fork, Mcm displays marked processivity, expected for a replicative helicase. These findings lead us to propose that selective activation by stretches of thymine sequences of a fraction of Mcm helicases loaded onto chromatin may be the determinant for selection of initiation sites on mammalian genomes.

In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast.

The Williams Syndrome Transcription Factor (WSTF), the product of the WBSCR9 gene, is invariably deleted in the haploinsufficiency Williams-Beuren Syndrome. Along with the nucleosome-dependent ATPase ISWI, WSTF forms a novel chromatin remodeling complex, WICH (WSTF-ISWI chromatin remodeling complex), which is conserved in vertebrates. The WICH complex was purified to homogeneity from Xenopus egg extract and was found to contain only WSTF and ISWI. In mouse cells, WSTF interacts with the SNF2H isoform of ISWI. WSTF accumulates in pericentric heterochromatin coincident with the replication of these structures, suggesting a role for WSTF in the replication of heterochromatin. Such a role is supported by the in vitro activity of both the mouse and frog WICH complexes: they are involved in the assembly of regular spaced nucleosomal arrays. In contrast to the related ISWI-interacting protein ACF1/WCRF180, WSTF binds stably to mitotic chromosomes. As dysfunction of other chromatin remodeling factors often has severe effects on development, haploinsufficiency of WSTF may explain some of the phenotypes associated with this disease.

Chromosome replication in Escherichia coli is initiated by the DnaA protein. Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process. Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation. In vitro studies have shown that SeqA inhibits oriC-dependent replication. Here we show by KMnO(4) probing that SeqA inhibits open complex formation. The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids. SeqA was found to restrain the negative supercoils of the oriC plasmid. In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively. It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids. SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.

In vivo studies suggest that the Escherichia coli SeqA protein modulates replication initiation in two ways: by delaying initiation and by sequestering newly replicated origins from undergoing re-replication. As a first approach towards understanding the biochemical bases for these effects, we have examined the effects of purified SeqA protein on replication reactions performed in vitro on an oriC plasmid. Our results demonstrate that SeqA directly affects the biochemical events occurring at oriC. First, SeqA inhibits formation of the pre-priming complex. Secondly, SeqA can inhibit replication from an established pre-priming complex, without disrupting the complex. Thirdly, SeqA alters the dependence of the replication system on DnaA protein concentration, stimulating replication at low concentrations of DnaA. Our data suggest that SeqA participates in the assembly of initiation-competent complexes at oriC and, at a later stage, influences the behaviour of these complexes.

The origin decision point (ODP) was originally identified as a distinct point during G1-phase when Chinese hamster ovary (CHO) cell nuclei experience a transition that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. Passage of cells through the ODP requires a mitogen-independent protein kinase that is activated prior to restriction point control. Here we show that inhibition of an early G1-phase protein kinase pathway by the addition of 2-aminopurine (2-AP) prior to the ODP arrests CHO cells in G1-phase. Transformation with simian virus 40 (SV40) abrogated this arrest point, resulting in the entry of cultured cells into S-phase in the presence of 2-AP and a disruption of the normal pattern of initiation sites at the DHFR locus. Cells treated with 2-AP after the ODP initiated replication specifically within the DHFR origin locus. Transient exposure of transformed cells to 2-AP during the ODP transition also disrupted origin choice, whereas non-transformed cells arrested in G1-phase and then passed through a delayed ODP after removal of 2-AP from the medium. We conclude that mammalian cells have many potential sites at which they can initiate replication. Normally, events occurring during the early G1-phase ODP transition determine which of these sites will be the preferred initiation site. However, if chromatin is exposed to S-phase-promoting factors prior to this transition, mammalian cells, like Xenopus and Drosophila embryos, can initiate replication without origin specification.