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Effect of mini-chromosome maintenance protein 2 deficiency on the locations of DNA replication origins

ABSTRACT: Mini-chromosome maintenance (MCM) proteins are loaded to chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10 and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more Mcm2-7 genes which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene rich locations.

ORGANISM(S): Mus musculus

PROVIDER: GSE64933 | GEO | 2015/01/14

REPOSITORIES: GEO

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Effect of mini-chromosome maintenance protein 2 deficiency on the locations of DNA replication origins

Project description:Mini-chromosome maintenance (MCM) proteins are loaded to chromatin during G1-phase and define potential locations of DNA replication initiation. MCM protein deficiency results in genome instability and high rates of cancer in mouse models. Here we develop a method of nascent strand capture and release and show that MCM2 deficiency reduces DNA replication initiation in gene rich regions of the genome. DNA structural properties are shown to correlate with sequence motifs associated with replication origins and with locations that are preferentially affected by MCM2 deficiency. Reduced nascent strand density correlates with sites of recurrent focal CNVs in tumors arising in MCM2-deficient mice, consistent with a direct relationship between sites of reduced DNA replication initiation and genetic damage. Between 10 and 90% of human tumors, depending on type, carry heterozygous loss or mutation of one or more Mcm2-7 genes which is expected to compromise DNA replication origin licensing and result in elevated rates of genome damage at a subset of gene rich locations. Mcm2 deficient vs. wild type

2015-01-14 | E-GEOD-64933 | biostudies-arrayexpress

SETD3-mediated histidine methylation of MCM7 regulates DNA replication by facilitating chromatin loading of MCM

Project description:The minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during G1/S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide deeper understanding towards this event. Here, we uncover that the histidine methyltransferase SETD3 promotes DNA replication in an enzymatic activity dependent manner. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Mechanistically, biochemical experiments reveal that SETD3 binds MCM mainly during G1/S phase, which is required for CDT1-mediated chromatin loading of MCM. The MCM loading relies on the histidine-459 methylation (H459me) on MCM7 that is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.

2024-03-12 | GSE243937 | GEO

Genome-wide expression analysis of human hTert immortalized fibroblasts after donwregulation of MCM7

Project description:Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

2018-10-20 | GSE120767 | GEO

Genome-wide expression analysis of human hTert immortalized fibroblasts after downregulation of MCM2

2018-10-20 | GSE120766 | GEO

A new MCM modification cycle regulates DNA replication initiation

Project description:The hexameric DNA helicase MCM (Mcm2-7) is a central regulatory target in eukaryotic replication. Chromatin-bound MCM is kept inactive during G1 phase and subsequently activated in S phase to initiate replication. During this transition, the only known chemical change on the Mcm2-7 proteins is the gain of multi-site phosphorylation that promotes recruitment of co-factors. As replication initiation is tied intimately to multiple biological cues, additional changes on these proteins can provide a second regulatory point. Here we describe a new MCM modification cycle mediated by SUMO that enables a negative regulation of replication initiation. We show that all MCM subunits undergo sumoylation upon loading at origins in G1 phase prior to MCM phosphorylation. Then bulk MCM sumoylation is lost as MCM phosphorylation rises. The pattern of MCM sumoylation suggests a negative role in replication. Indeed, increasing MCM sumoylation delays genome-wide replication initiation. Mechanistically, this is partly due to enhancing the recruitment of a conserved phosphatase that delays MCM phosphorylation and activation. By revealing a new MCM modification cycle and its role in replication, our findings suggest a new model, in which MCM sumoylation counterbalances kinase-based regulation to ensure accurate control of replication initiation.

2016-02-01 | GSE70407 | GEO

DNA Polymerase Delta Governs Parental Histone Transfer to DNA Replication Lagging Strand

Project description:Chromatin replication is intricately intertwined with DNA replication, and the recycling of parental histones is essential for epigenetic inheritance. The transfer of parental histones to both the DNA replication leading and lagging strands involves two distinct pathways: the leading strand utilizes DNA polymerase ε subunits Dpb3/Dpb4, while the lagging strand is facilitated by the MCM helicase subunit Mcm2. However, the process by which Mcm2, moving along the leading strand, facilitates the transfer of parental histones to the lagging strand remains unclear. Our study reveals that the deletion of Pol32, a non-essential subunit of major lagging-strand DNA polymerase δ, results in a predominant transfer of parental histone H3-H4 to the replication leading strand. Biochemical analyses further demonstrate that Pol32 can bind histone H3-H4 both in vivo and in vitro. The interaction of Pol32 with parental histone H3-H4 is disrupted by the mutation of Mcm2's histone H3-H4 binding domain. In conclusion, our findings identify the DNA polymerase delta subunit Pol32 as a novel key histone chaperone downstream of Mcm2, mediating the transfer of parental histones to the lagging strand during DNA replication.

2024-04-24 | GSE252049 | GEO

Destabilization of the MiniChromosome Maintenance (MCM) complex modulates the cellular response to DNA double strand breaks induced by Etoposide.

Project description:DNA replication during S phase involves thousands of replication forks that must be coordinated to ensure that every DNA section is only replicated once. The minichromosome maintenance proteins, MCM2 to MCM7, form a heteromeric DNA helicase required for both initiation of DNA replication and elongation of DNA replication. Although only two DNA helicase activities are required to establish a bidirectional replication fork from each origin of replication, a large excess of MCM complexes is loaded and distributed along the chromatin. The function of the additional MCM complexes is not well understood, as most of them are displaced from the DNA during S-phase, apparently without playing an active role in DNA replication. DNA damage response (DDR) kinases activated by stalled forks prevent the replication machinery from being activated, indicating a tight relationship between DDR and DNA replication. To investigate the role of MCM proteins in the cellular response to DNA damage, we used shRNA targeting MCM2 or MCM3 to determine the impact of the reduction of the MCM complex. The alteration of MCM proteins induced a change in the activation of important factors of the DDR in response to Etoposide treatment. The phosphorylation of γ-H2AX, CHK1 and CHK2 is affected following DNA damage induced by treatment with Etoposide without affecting cell viability. Using assays measuring homologous recombination (HR) and non-homologous end-joining (NHEJ), we have identified a decrease of HR, but not NHEJ, associated with a decrease of the MCM complex demonstrating a role for the MCM complex in homologous recombination.

2019-11-12 | PXD009539 | Pride

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy

Project description:During routine genome duplication, many potential replication origins remain inactive, or dormant. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that can be distinguished from baseline (i.e. ordinarily active) origins by their preferential association with MCM2, a component of the replicative helicase, phosphorylated on serine 108 (pS108-MCM2). pS108-MCM2 is a substrate of the ATR kinase, which is recruited to chromatin via an interaction with hyperacetylated TOPBP1 in cells undergoing replication stress or in cells devoid of SIRT1 deacetylase activity. In turn, S108-MCM2 phosphorylation enhances a second, DDK-dependent, S139-MCM2 phosphorylation, which triggers initiation of DNA replication at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational modifications on the MCM helicase that reflect a balance between SIRT1 activity and ATR signaling.

| MSV000088134 | MassIVE

Convergence of SIRT1 and ATR signaling to modulate replication origin dormancy

Project description:During routine genome duplication, many potential replication origins remain inactive, or 'dormant'. Such origin dormancy is achieved, in part, by an interaction with the metabolic sensor SIRT1 deacetylase. We report here that dormant origins are a group of consistent, pre-determined genomic sequences that can be distinguished from baseline (i.e. ordinarily active) origins by their preferential association with MCM2, a component of the replicative helicase, phosphorylated on serine 108 (pS108-MCM2). pS108-MCM2 is a substrate of the ATR kinase, which is recruited to chromatin via an interaction with hyperacetylated TOPBP1 in cells undergoing replication stress or in cells devoid of SIRT1 deacetylase activity. In turn, S108-MCM2 phosphorylation enhances a second, DDK-dependent, S139-MCM2 phosphorylation, which triggers initiation of DNA replication at dormant origins. These observations suggest that replication origin dormancy and activation are regulated by distinct post-translational modifications on the MCM helicase that reflect a balance between SIRT1 activity and ATR signaling.

2022-05-09 | GSE184353 | GEO

H4S47 O-GlcNAcylation regulates the activation of mammalian replication origins

Project description:The transmission and maintenance of genetic information in eukaryotic cells rely on the faithful duplication of the entire genome. In each round of division, excessive replication origiNS are licensed, while only a fraction is activated to give rise to bi-directional replication forks travelling in the context of chromatin. However, it remaiNS elusive how eukaryotic replication origiNS are selectively activated. Here we demoNStrate that O-GlcNAc traNSferase (OGT) enhances replication initiation by catalysing H4S47 O-GlcNAcylation. Mutation of H4S47 impairs DBF4-dependent protein kinase (DDK) recruitment on chromatin, causing compromised phosphorylation of replicative helicase mini-chromosome maintenance (MCM) complex. Meanwhile, our short nascent-strand sequencing (SNS-seq) confirms the important role of H4S47 O-GlcNAcylation in origin activation. Together, H4S47 O-GlcNAcylation directs origin activation through facilitating MCM phosphorylation, and this may shed light on the spatial and temporal control of DNA replication by the dynamically changing chromatin environment.

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