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Project description:A very important feature of replication initiation proteins is the ability to bind to DNA via a characteristic domain. Usually, one or two DNA-binding domains can be distinguished within each initiator. In this work, we specified a new family of replication initiation proteins (the TrfA-like protein family) with unique domain compositions that are important for interactions with DNA. Using phylogenetic analysis and structure prediction methods simultaneously with biochemical assays, we demonstrate that in the replication initiator of the broad-host-range plasmid RK2, in addition to two winged helix domains, a third domain that interacts with DNA can be described. Mass spectrometric analysis followed by site-directed mutagenesis and in vitro and in vivo analysis of TrfA variants showed that DNA binding by all three domains of TrfA is important for stable nucleoprotein complex formation and the replication activity of the initiator.
2021-09-08 | PXD013286 | Pride
Project description:The replication initiation proteins interact dsDNA located at replication origin and ssDNA of DNA unwinding element (DUE), formed as a result of the destabilization of the double-stranded helix of AT-rich origin region. It is a critical step in the DNA replication initiation; however, the structure of nucleoprotein complex involving initiator protein, dsDNA and/or ssDNA is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), structural and bioinformatic analysis, we defined amino acid residues in plasmid Rep proteins, TrfA and RepE, that are essential for interaction with ssDNA. The study of Rep mutant proteins containing single amino acid substitutions affecting DNA interaction reveals the importance of Rep-ssDNA complexes formation for a dsDNA melting at DUE. Furthermore, the crystal structures obtained for complex of RepE protein with DUE ssDNA, and, RepE complexed with both DUE ssDNA and dsDNA containing RepE specific binding site (iteron) revealed that the plasmid initiator can not only bind iterons and ssDNA DUE separately but also can form a tripartite nucleoprotein complex bringing together specific sequences of replication origin. The presented data strongly supports the loop-back model in which a replication initiator molecule interacts with dsDNA and ssDNA.
2024-01-26 | PXD032398 | Pride
Project description:Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription. Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription.
2019-12-03 | GSE127679 | GEO
Project description:Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription. Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription.
2019-03-11 | GSE127678 | GEO
Project description:Loss of DNA replication control is a potent initiator of gene amplification
| PRJNA127435 | ENA
Project description: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. Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).
2013-06-06 | E-GEOD-41982 | biostudies-arrayexpress
Project description:The chromatin-based rules governing the selection and activation of replication origins remain to be elucidated. It is believed that DNA replication initiates from open chromatin domains, thus replication origins residing in regulatory elements that are located at open and active chromatin. However, we report here that lysine specific demethylase 1 (LSD1), which biochemically catalyzes H3K4me1/2 demethylation to favor chromatin condensation, interacts with the DNA replication machinery. We found that LSD1 level peaks in early S phase. We demonstrated that LSD1 promotes DNA replication by facilitating origin firing in euchromatic regions and through regulating replication timing. Indeed, euchromatic zones enriched in H3K4me2 are the preferred sites for pre-RC binding in early replication. Remarkably, LSD1 deficiency leads to a genome-wide switch from early to late in replication timing. We showed that LSD1-promoted DNA replication is mechanistically linked to the loading of TICRR (TopBP1-Interacting Checkpoint and Replication Regulator) onto the pre-RC and subsequent recruitment of the initiator Cdc45 during origin firing. Together, these results reveal an unexpected role for LSD1 in euchromatic origin firing and replication timing.
2022-03-07 | GSE157469 | GEO
Project description:Replication and segregation are the two main processes that maintain chromosomes in growing cells. In bacteria, replication and transcription have been proposed to provide motive force in chromosome segregation. Recently, ParB2, a segregation protein, encoded by V. cholerae chromosome II (chrII) was also found to influence chrII replication. V. cholerae chrII replication is primarily controlled by its specific initiator protein, RctB. Here, we have screened for ParB2 and RctB binding sites using a genome-wide DNA binding analysis (ChIP-chip). We report the identification of a new region containing additional RctB and ParB2 binding sites, and suggest the mechanisms to coordinate replication initiation with segregation of chromosomes.
2015-09-14 | GSE72978 | GEO
Project description: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.
2013-06-06 | GSE41982 | GEO
Project description:Polycomb proteins link Proliferation with DNA Replication
| PRJNA210421 | ENA