ORC binds and remodels nucleosomes to specify MCM loading onto DNA

ABSTRACTSaccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection

Chromatin loading of MCM hexamers is associated with di-/tri-methylation of histone H4K20 toward S phase entry

Replication of genomic DNA is a key step in initiating cell proliferation. Loading hexameric complexes of minichromosome maintenance (MCM) helicase on DNA replication origins during the G1 phase is essential in initiating DNA replication. Here, we show that stepwise loading of two hexamer complexes of MCM occurs during G1 progression in human cells. This transition from the single-to-double hexamer was associated with levels of methylation at lysine 20 of histone H4 (H4K20). A single hexamer of MCM complexes was loaded at the replication origins with the presence of H4K20 monomethylation (H4K20me1) in the early G1 phase, then another single hexamer was recruited to form a double hexamer later in G1 as H4K20me1 was converted to di-/tri-methylation (H4K20me2/me3). Under non-proliferating conditions, cells stay halted at the single-hexamer state in the presence of H4K20me1. We propose that the single-hexamer state on chromatin is a limiting step in making the proliferation-quiescence decision.

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions (‘multiple fission’). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom.

DNA replication origins retain mobile licensing proteins

DNA replication in eukaryotes initiates at many origins distributed across each chromosome. Origins are bound by the origin recognition complex (ORC), which, with Cdc6 and Cdt1, recruits and loads the Mcm2-7 (MCM) helicase as an inactive double hexamer during G1 phase. The replisome assembles at the activated helicase in S phase. Although the outline of replisome assembly is understood, little is known about the dynamics of individual proteins on DNA and how these contribute to proper complex formation. Here we show, using single-molecule optical trapping and confocal microscopy, that yeast ORC is a mobile protein that diffuses rapidly along DNA. Origin recognition halts this search process. Recruitment of MCM molecules in an ORC- and Cdc6-dependent fashion results in slow-moving ORC-MCM intermediates and MCMs that rapidly scan the DNA. Following ATP hydrolysis, salt-stable loading of MCM single and double hexamers was seen, both of which exhibit salt-dependent mobility. Our results demonstrate that effective helicase loading relies on an interplay between protein diffusion and origin recognition, and suggest that MCM is stably loaded onto DNA in multiple forms.

Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

Conservation and divergence of DNA replication control in Chlamydomonas reinhardtii

ABSTRACTWe recently isolated temperature-sensitive cell cycle mutants in Chlamydomonas reinhardtii for which the causative mutations were located in genes annotated for potential involvement in DNA replication. Chlamydomonas has a very long G1 period during which cells grow up to ~10-fold without division, followed by rapid cycles of DNA replication and mitosis (‘multiple fission’). All of the candidate DNA replication mutants tested were defective in completion of the first round of DNA replication, and also failed to produce mitotic spindles. For a subset of the mutants, we rescued temperature-sensitive lethality with tagged transgenes and used the resulting strains to analyze abundance and localization control of the tagged protein. All of the DNA replication proteins tested were essentially undetectable until late G1, accumulated during the period of multiple fission and then were degraded as cells completed their terminal divisions. MCM4 and MCM6 were localized to the nucleus during the division cycle except for transient cytoplasmic localization during mitosis. CDC45 showed strict protein location to the nucleus and co-localized to spindles during mitosis. In contrast, CDC6 was detected in the nucleus only transiently during early divisions within the overall multiple fission cycle. Cdc6 protein levels were very low, but increased upon treatment with MG132, a proteasome inhibitor. We also tested if these DNA replication proteins are regulated by cyclin dependent kinase (CDK). There are two main CDKs in Chlamydomonas, CDKA1 and CDKB1. We found that CDC6 protein level was severely reduced in a cdka1 mutant, but not in a cdkb1 mutant. MG132 did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 upregulates CDC6 at the transcription level. Since MCM4, MCM6 and CDC6 were all essentially undetectable during the long G1 before DNA replication cycles began, we speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. These results provide a microbial framework for approaching replication control in the plant kingdom.

The GINS complex is required for the survival of rapidly proliferating retinal and tectal progenitor cells during zebrafish development

Efficient and accurate DNA replication is particularly critical in stem and progenitor cells for successful proliferation and survival. The replisome, an amalgam of protein complexes, is responsible for binding potential origins of replication, unwinding the double helix, and then synthesizing complimentary strands of DNA. According to current models, the initial steps of DNA unwinding and opening are facilitated by the CMG complex, which is composed of a GINS heterotetramer that connects Cdc45 with the mini-chromosome maintenance (Mcm) helicase. In this work, we provide evidence that in the absence of GINS function DNA replication is cell autonomously impaired, and we also show that gins1 and gins2 mutants exhibit elevated levels of apoptosis restricted to actively proliferating regions of the central nervous system (CNS). Intriguingly, our results also suggest that the rapid cell cycles during early embryonic development in zebrafish may not require the function of the GINS complex as neither Gins1 nor Gins2 seem to be present during these stages.

Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.

Multivalent interaction of ESCO2 with the replication machinery is required for cohesion

The tethering together of sister chromatids by the cohesin complex ensures their accurate alignment and segregation during cell division. In vertebrates, the establishment of cohesion between sister chromatids requires the activity of the ESCO2 acetyltransferase, which modifies the Smc3 subunit of cohesin. It was shown recently that ESCO2 promotes cohesion through interaction with the MCM replicative helicase. However, ESCO2 does not significantly colocalize with the MCM helicase, suggesting there may be additional interactions that are important for ESCO2 function. Here we show that ESCO2 is recruited to replication factories, the sites of DNA replication. We show that ESCO2 contains multiple conserved PCNA-interaction motifs in its N-terminus, and that each of these motifs are essential to ESCO2’s ability to establish sister chromatid cohesion. We propose that multiple PCNA interaction motifs embedded in a largely flexible and disordered region of the protein underlie the ability of ESCO2 to establish cohesion between sister chromatids precisely as they are born during DNA replication.SummaryCohesin modification by the ESCO2 acetyltransferase is required for cohesion between sister chromatids. Here we identify multiple motifs in ESCO2 that mediate its interaction with the replication processivity factor PCNA, and show that their mutation abrogates the ability of ESCO2 to ensure cohesion.