ERCC6L2 mitigates replication stress and promotes centromere stability [Nascent_DNA_seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE226154 | GEO | 2023/04/10
REPOSITORIES: GEO
Similar Datasets
Project description:Structurally complex genomic regions, such as centromeres, are inherently difficult to duplicate. The mechanism that underlies centromere inheritance is not well understood, and one of the key questions relates to the reassembly of centromeric chromatin following DNA replication. Here we define the SNF2 ATPase ERCC6L2 as a key regulator of this process. ERCC6L2 accumulates at centromeres and promotes efficient deposition of core centromeric factors. Our genomic analyses show that ERCC6L2 deficiency erodes centromeric chromatin, leading to unrestrained replication of centromeric DNA. We also establish that, beyond centromeres, ERCC6L2 facilitates replication at genomic repeats and non-canonical DNA structures. Notably, ERCC6L2 interacts with the major DNA replication factor PCNA through an atypical peptide, presented here as a co-crystal structure. Finally, we examine ERCC6L2 activities at DNA breaks, and show that it acts to restrict end resection independently of the 53BP1-REV7-Shieldin complex. Our observations allow us to propose a mechanistic model of ERCC6L2 activity, which reconciles its seemingly distinct functions in DNA repair and DNA replication. Together, these findings provide a new molecular context for studies linking ERCC6L2 to human disease.
2023-04-10 | GSE226152 | GEO
Project description:53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin—a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)— as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway.
2018-07-26 | PXD009650 | Pride
Project description:In order to address the replication timing of the centromeres in yeast Candida albicans, we have determined at high temporal and spatial resolution its genome-wide DNA replication timing profile. This was done by sorting S- and G1-phase cells by FACS and hybridizing DNA from both fractions onto genomic tiling arrays. Data was normalized, organized by chromosomal positition, and smoothed. Replication timing is inferred from the DNA copy number along the chromosome with data peaks corresponding to predicted origins of DNA replication. We observe that the centromeric loci coincide with replication origins that are the first to replicate on each chromosome. We further analyzed replication timing in strain 9929s, in which the centromere of chromosome 5 was deleted and a neocentromere has formed at a distant location. A replication origin, again the first to replicate, appeared at the site of neocentromere formation. We conclude that centromeres cause their own early and distinct DNA replication timing.
2010-08-31 | GSE17963 | GEO
Project description:Centromeres play an essential role in cell division by specifying the site of kinetochore formation on each chromosome so that chromosomes can attach to the mitotic spindle for segregation. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). Dividing cells maintain the centromere by depositing new CENP-A each cell cycle to replenish CENP-A diluted by replication. The CENP-A nucleosome serves as the primary signal to the machinery responsible for its replenishment. Vertebrate centromeres are frequently built on repetitive sequences organized in tandem arrays. Repetitive centromeric DNA has been suggested to play a role in centromere maintenance and in de novo centromere formation, but this has been difficult to dissect because of the difficulty in manipulating centromere in cells. Extracts from Xenopus laevis eggs are able to assemble centromeres and kinetochores in vitro and thus provide a useful system for studying the role of centromeric DNA in centromere formation. However centromeric sequences in X. laevis have not been extensively characterized.. In this study we characterize repeat sequences found at X. laevis centromeres. We utilize a k-mer based approach in order to uncover the previously unknown diversity of X. laevis centromeric sequences. We validate centromere localization of repeat sequences by in situ hybridization and identify the location of the centromeric repetitive array on each chromosome by mapping the distribution of centromere enriched k-mers on the Xenopus genome. Our identification of X. laevis centromere sequences enables previously unapproachable genomic studies of centromeres. The k-mer based approach that we used to investigate centromeric repetitive DNA is suitable for the analysis of other repetitive sequences found across the genome or the study of repeats in other organisms.
2020-06-24 | GSE153058 | GEO
Project description:MAD2L2 (also known as REV7) functions in a diverse range of processes. It facilitates DNA lesion bypass through translesion synthesis (TLS), aids in interstrand crosslink repair and contributes to timely mitotic progression. Moreover, MAD2L2 plays an important role in DNA repair at DNA double strand breaks (DSBs) and uncapped telomeres. As the first identified member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice between non-homologous end-joining (NHEJ) and homologous recombination (HR) by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization surface, HORMA domain containing protein MAD2L2 has the extraordinary ability to wrap its C-terminal tail around an interacting peptide motif in SHLD3, thereby likely creating a very stable complex. We show that appropriate function of MAD2L2 in DNA repair as a member of the shieldin complex requires it to form a dimer that is mediated by SHLD2 and accelerates MAD2L2-SHLD3 interaction. Dimerization defective MAD2L2 impairs assembly of shieldin and fails to promote NHEJ at telomeres and during immunoglobulin class switch recombination (CSR). Moreover, dimerization of MAD2L2, along with the presence of SHLD3, allows the shieldin complex to interact with the AAA+ family ATPase TRIP13, known to drive topological switches in HORMA domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.
2021-09-21 | PXD026912 | Pride
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:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
2018-11-29 | GSE106491 | GEO
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
2018-11-29 | GSE106492 | GEO
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
2018-11-29 | GSE119922 | GEO
Project description:Centromeres are the regions of eukaryotic chromosomes where kinetochores are assembled and direct the correct segregation of chromosomes. Active centromeres are defined by presence of nucleosomes containing CENP-A, a histone H3 variant, which alone is sufficient to direct kinetochore assembly. Once assembled at a location CENP-A chromatin and the kinetochore is maintained at that location though a positive feedback loop where kinetochore proteins recruited by CENP-A promote deposition of new CENP-A following replication. Although CENP-A chromatin itself is a heritable entity, it is normally associated with specific sequences such as human alpha satellite arrays. Such analyses suggest that properties of centromeric DNA itself may favour assembly of CENP-A rather than H3 nucleosomes. To investigate the innate properties of centromeric DNA we have examined histone dynamics on this DNA assembled in CENP-A chromatin at endogenous centromeres and when assembled only in H3 chromatin at an ectopic location. We demonstrate that H3 occupancy on centromeric DNA is innately low while H3 turnover is high. Moreover, even at an ectopic location centromeric DNA programs H3 deposition in S phase and its eviction during G2 when CENP-A is otherwise deposited. G2 accumulation of RNAPII on centromeric DNA during G2 is consistent with transcription-coupled destabilisation of H3 nucleosomes to favour CENP-A deposition.
2019-03-04 | GSE112454 | GEO