The Requirement of Yeast Replication Origins for Pre-Replication Complex Proteins is Modulated by Transcription (original) (raw)
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Genome-wide localization of pre-RC sites and identification of replication origins in fission yeast
DNA replication of eukaryotic chromosomes initiates at a number of discrete loci, called replication origins. Distribution and regulation of origins are important for complete duplication of the genome. Here, we determined locations of Orc1 and Mcm6, components of pre-replicative complex (pre-RC), on the whole genome of Schizosaccharomyces pombe using a high-resolution tiling array. Pre-RC sites were identified in 460 intergenic regions, where Orc1 and Mcm6 colocalized. By mapping of 5-bromo-2 0deoxyuridine (BrdU)-incorporated DNA in the presence of hydroxyurea (HU), 307 pre-RC sites were identified as earlyfiring origins. In contrast, 153 pre-RC sites without BrdU incorporation were considered to be late and/or inefficient origins. Inactivation of replication checkpoint by Cds1 deletion resulted in BrdU incorporation with HU specifically at the late origins. Early and late origins tend to distribute separately in large chromosome regions. Interestingly, pericentromeric heterochromatin and the silent mating-type locus replicated in the presence of HU, whereas the inner centromere or subtelomeric heterochromatin did not. Notably, MCM did not bind to inner centromeres where origin recognition complex was located. Thus, replication is differentially regulated in chromosome domains.
Journal of Biological Chemistry, 2013
Background: MCM-BP is a novel binding partner of the MCM complex; the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Results: Genetic analysis showed that mcb1 ts mutants exercise defective regulation of prereplicative MCM complex formation during DNA replication. Conclusion: Mcb1 regulates MCM function during prereplicative complex formation in DNA replication. Significance: This study presents the first evidence of MCM-BP function during prereplicative complex formation. The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1 ؉ , two temperature-sensitive mcb1 gene mutants (mcb1 ts) were isolated. Extensive genetic analysis showed that the mcb1 ts mutants were suppressed by a mcm5 ؉ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1 ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1 ts mutants. Furthermore, the mcb1 ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. Genome integrity depends on successful and faithful DNA replication, which relies on the concerted activity of multiple replication proteins. The temporal and spatial regulation of DNA replication and the cell cycle control system ensure a single round of replication of chromosome DNA during every cell cycle. The initiation of DNA replication in all eukaryotes involves the assembly of a prereplicative complex (pre-RC) 2 at the replication origins in G 1 phase and the subsequent activation of the pre-RC to the preinitiation complex (pre-IC) at the onset of S-phase. The replication origins are recognized by the origin recognition complex and become a platform for the recruitment of Cdc6 (called Cdc18 in fission yeast) and Cdt1bound double hexamers of the minichromosome maintenance (MCM) complex to form the pre-RC (reviewed in Ref. 1). Upon entry into S-phase, this complex is activated by the S-phasespecific kinase, Dbf4/Drf1-dependent kinase. The Mcm2-7 complex serves as a platform to recruit Cdc45 and GINS, thereby converting the pre-RC into the pre-IC. Dbf4/Drf1-dependent kinase phosphorylates several of the Mcm2-7 proteins and triggers the recruitment of Cdc45 and GINS to form the Cdc45⅐MCM⅐GINS complex. Cdc45⅐MCM⅐GINS complex formation then induces the helicase activity of the Mcm2-7 complex, promoting the unwinding of the double-stranded DNA at the origin of DNA replication (reviewed in Ref. 2). The Mcm2-7 hexamer complex is an evolutionarily conserved DNA helicase, which is essential for both the initiation of chromosome DNA replication and elongation (3, 4). Mcm2-7 * This work was supported by a grant-in-aid for scientific research from the Japan Society for the Promotion of Science and the Support Project to Assist Private Universities in Developing Bases for Research by the Ministry of Education, Culture, Sports, Science, and Technology (to K. T.). □ S This article contains supplemental Tables S1 and S2 and Figs. S1-S7.
Relicensing of Transcriptionally Inactivated Replication Origins in Budding Yeast
Journal of Biological Chemistry, 2010
DNA replication origins are licensed in early G 1 phase of the cell cycle where the origin recognition complex (ORC) recruits the minichromosome maintenance (MCM) helicase to origins. These pre-replicative complexes (pre-RCs) remain inactive until replication is initiated in the S phase. However, transcriptional activity in the regions of origins can eliminate their functionality by displacing the components of pre-RC from DNA. We analyzed genome-wide data of mRNA and cryptic unstable transcripts in the context of locations of replication origins in yeast genome and found that at least one-third of the origins are transcribed and therefore might be inactivated by transcription. When investigating the fate of transcriptionally inactivated origins, we found that replication origins were repetitively licensed in G 1 to reestablish their functionality after transcription. We propose that reloading of pre-RC components in G 1 might be utilized for the maintenance of sufficient number of competent origins for efficient initiation of DNA replication in S phase.
Proceedings of the National Academy of Sciences, 1992
Saccharomyces cerevisiae cells containing mutations in the cell-division-cycle gene CDC46 arrest with a large bud and a single nucleus with unreplicated DNA at the non-permissive temperature. This G1/S arrest, together with the increased rates of mitotic chromosome loss and recombination phenotype, suggests that these mutants are defective in DNA replication. The subcellular localization of the CDC46 protein changes with the cell cycle; it is nuclear between the end of M phase and the G1/S transition but is cytoplasmic in other phases of the cell cycle. Here we show that CDC46 is identical to MCM5, based on complementation analysis of the mcm5-1 and cdc46-1 alleles, complementation of the minichromosome maintenance defect of mcm5-1 by CDC46, and the genetic linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 also show a minichromosome maintenance defect thought to be associated with DNA replication initiation at autonomously replicating sequences. Taken together, these obse...
EMBO reports, 2012
To elucidate the role of the chromatin environment in the regulation of replication origin activation, autonomously replicating sequences were inserted into identical locations in the budding yeast genome and their activation times in S phase determined. Chromatin-dependent origins adopt to the firing time of the surrounding locus. In contrast, the origins containing two binding sites for Forkhead transcription factors are activated early in the S phase regardless of their location in the genome. Our results also show that genuinely late-replicating parts of the genome can be converted into early-replicating loci by insertion of a chromatin-independent early replication origin, ARS607, whereas insertion of two Forkhead-binding sites is not sufficient for conversion.
Initiation of DNA Replication from Non-Canonical Sites on an Origin-Depleted Chromosome
PLoS ONE, 2014
Eukaryotic DNA replication initiates from multiple sites on each chromosome called replication origins (origins). In the budding yeast Saccharomyces cerevisiae, origins are defined at discrete sites. Regular spacing and diverse firing characteristics of origins are thought to be required for efficient completion of replication, especially in the presence of replication stress. However, a S. cerevisiae chromosome III harboring multiple origin deletions has been reported to replicate relatively normally, and yet how an origin-deficient chromosome could accomplish successful replication remains unknown. To address this issue, we deleted seven wellcharacterized origins from chromosome VI, and found that these deletions do not cause gross growth defects even in the presence of replication inhibitors. We demonstrated that the origin deletions do cause a strong decrease in the binding of the origin recognition complex. Unexpectedly, replication profiling of this chromosome showed that DNA replication initiates from non-canonical loci around deleted origins in yeast. These results suggest that replication initiation can be unexpectedly flexible in this organism.
Genome-wide characterization of fission yeast DNA replication origins
The EMBO Journal, 2006
Eukaryotic DNA replication is initiated from multiple origins of replication, but little is known about the global regulation of origins throughout the genome or in different types of cell cycles. Here, we identify 401 strong origins and 503 putative weaker origins spaced in total every 14 kb throughout the genome of the fission yeast Schizosaccharomyces pombe. The same origins are used during premeiotic and mitotic S-phases. We found that few origins fire late in mitotic S-phase and that activating the Rad3 dependent S-phase checkpoint by inhibiting DNA replication had little effect on which origins were fired. A genome-wide analysis of eukaryotic origin efficiencies showed that efficiency was variable, with large chromosomal domains enriched for efficient or inefficient origins. Average efficiency is twice as high during mitosis compared with meiosis, which can account for their different S-phase lengths. We conclude that there is a continuum of origin efficiency and that there is differential origin activity in the mitotic and meiotic cell cycles.
Recent advances in the genome-wide study of DNA replication origins in yeast
Frontiers in microbiology, 2015
DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches tha...
Genes & development, 2015
Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficienc...