Incongruent end structures of leading and lagging telomeres dictate the nature of end replication problem
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ABSTRACT:
PROVIDER: PRJNA1193986 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31596119_1.fastq.gz | Fastqsanger.gz | |||
| SRR31596119_2.fastq.gz | Fastqsanger.gz | |||
| SRR31596120_1.fastq.gz | Fastqsanger.gz | |||
| SRR31596120_2.fastq.gz | Fastqsanger.gz | |||
| SRR31596121_1.fastq.gz | Fastqsanger.gz |
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Project description:The end replication problem refers to the incomplete replication of parental DNA at telomeres, a process whose molecular depiction is hampered by the complex nature of telomere ends. Here we recapitulate this process using a synthetic de novo telomere in yeast and delineate distinct molecular fates of telomere ends in vivo. We show that the lagging strand telomere carries a ~10 nt 3’ overhang, while the leading strand telomere has a Yku-protected blunt end, which is prevalent on native telomeres. Moreover, we find that RNase H2 is mainly responsible for the removal of the last RNA primer. In the absence of RNase H2 activity, RNA primer retention on the lagging strand telomere attenuates telomere erosion and delays senescence in telomerase-null cells. These findings highlight incongruent end structures on telomeres and clarify that the primary culprit behind end replication problem is the incompletely replicated lagging strand telomere.
2024-12-09 | GSE283459 | GEO
Project description:Incongruent end structures of leading and lagging telomeres dictate the nature of end replication problem
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Project description:The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length homeostasis. Previous studies addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a genetic screen in combination with high throughput sequencing to identify several amino acid positions in Rap1 that have a key role in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner with longer telomeres being prone to undergoing end fusions, while short telomeres are sufficiently protected. The protection of long telomeres requires their nuclear envelope attachment mediated by the Rap1-Bqt4 interaction. Our data demonstrates that longer telomeres pose an additional challenge for the maintenance of genome integrity and provides an explanation for a species-specific upper limit in telomere length.
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Project description:SDL-TOPO protocol was applied for the detection of lagging-strand in replicating budding yeast strain. Genomic DNA was extracted from cdc9 mutant strain, and served for library preparation with SDL-TOPO. To see the sensitivity of SDL-TOPO for the detection of lagging strand, various amounts of genomic DNA were used for the library preparation: 75 ng, 25 ng, 7.5 ng, 2.5 ng, 750 pg, 250 pg, 75 pg, and 25 pg. The prepared libraries were sequenced on Illumina iSeq100 with paired-end mode of 150x2. Only read 1 was served for mapping with bowtie2 against reference sequence sacSer3. The mapped reads were separated strand specifically, and read depths were visualized. The strand-biased mapping patterens were observed for libraries prepared from each amount of input DNA.
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Project description:We show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur around nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging strand polymerase processivity impacts both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of lagging strand synthesis. Our studies represent the first high-resolution analysis of eukaryotic Okazaki fragments in vivo, and establish a mechanistic link between the fundamental processes of DNA replication and chromatin assembly. 4 samples: replicate samples of wild-type and pol32 knockout
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Project description:In response to DNA replication stress, DNA replication checkpoint is activated to maintain fork stability, a process critical for maintenance of genome stability. However, how DNA replication checkpoint regulates replication forks remain elusive. Here we show that Rad53, a highly conserved replication checkpoint kinase, functions to couple leading and lagging strand DNA synthesis. In wild type cells under HU induced replication stress, synthesis of lagging strand, which contains ssDNA gaps, is comparable to leading strand DNA. In contrast, synthesis of lagging strand is much more than leading strand, and consequently, leading template ssDNA coated with ssDNA binding protein RPA was detected in rad53-1 mutant cells, suggesting that synthesis of leading strand and lagging strand DNA is uncoupled. Mechanistically, we show that replicative helicase MCM and leading strand DNA polymerase Pole move beyond actual DNA synthesis and that an increase in dNTP pools largely suppresses the uncoupled leading and lagging strand DNA synthesis. Our studies reveal an unexpected mechanism whereby Rad53 regulates replication fork stability.
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Project description:During classical non-homologous end joining (cNHEJ), DNA-dependent protein kinase (DNA-PK) encapsulates free DNA ends, forming a recruitment platform for downstream end-joining factors including Ligase 4 (LIG4). DNA-PK can also bind telomeres, but does not initiate cNHEJ at this position. How the end joining process is regulated in this context-specific manner is currently unclear. Here we show that the Shelterin components TRF2 and RAP1 form a complex with DNA-PK that directly represses its end joining function at telomeres. Biochemical experiments and cryo-electron microscopy reveal that when bound to TRF2, RAP1 establishes a network of interactions with KU and DNA that prevents DNA-PK from recruiting LIG4. In cells, RAP1 is redundant with the APOLLO nuclease in repressing cNHEJ at chromosome ends, demonstrating that the inhibition of DNA-PK prevents telomere fusions in parallel with overhang-dependent mechanisms. Our experiments show that the end joining function of DNA-PK is specifically and directly repressed at telomeres, describing a molecular mechanism for the maintenance of individual linear chromosomes in mammalian cells.
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Project description:Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres in vivo, as shown among other approaches by ChIP-seq analysis, and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting the other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.
2023-10-23 | GSE196661 | GEO
Project description:Telomeres, which locate at the end of chromosomes protect chromosomes from fusion and deterioration. This is essential for cells to maintain genome staibility. We applied RNA-seq, to identify the impact of Terc knockout on the expression of retrotransposons in embryonic stem cells.
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Project description:How parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications, are transferred to leading and lagging strands of DNA replication forks following DNA replication is an important question that remains not well understood. Here we show that DNA polymerase clamp PCNA and its partner involved in lagging strand DNA synthesis, Pol d, regulate parental histone transfer to lagging strands. Mutations at PCNA as well as at subunits of Pol d that impair the PCNA-Pol d interaction affect parental histone transfer to lagging strands, and this defect unlikely arises from their impacts on DNA synthesis. Moreover, Pol d interacts with H3-H4 in vitro. We suggest that the PCNA-Pol d complex, best known for its role in lagging strand DNA synthesis and DNA repair, couples lagging strand DNA synthesis to the transfer of the parental histones H3-H4 for the inheritance of chromatin structures following DNA replication and possibly DNA repair.
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