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A stress-induced error prone 3' flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival

ABSTRACT: A stress-induced error prone 3' flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival

PROVIDER: PRJNA741273 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14908070_1.fastq.gz	Fastqsanger.gz
	SRR14908070_2.fastq.gz	Fastqsanger.gz
	SRR14908071_1.fastq.gz	Fastqsanger.gz
	SRR14908071_2.fastq.gz	Fastqsanger.gz
	SRR14908072_1.fastq.gz	Fastqsanger.gz

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A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (WGS)

Project description:Faithful and efficient Okazaki fragment maturation is the key to maintain the genome integrity. In addition, this process is coupled with deposition of histone proteins. Therefore, functional deficiency in Okazaki fragment maturation may cause genome instablities as well and epigenetic alterations. We conduct WGS of WT and rad27 knockout yeast strains to define the DNA mutations caused by defective Okazaki fragment muturaton and conduct RNA-seq to define the changes in gene expression profiling due to defetive Okazaki fragment muturations..

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A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (RNA-seq)

2021-11-01 | GSE178873 | GEO

A stress-induced error prone 3' flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival

Project description:This SuperSeries is composed of the SubSeries listed below.

2021-11-01 | GSE178876 | GEO

A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (WGS)

Project description:A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (WGS)

| PRJNA741276 | ENA

A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (RNA-seq)

Project description:A stress-induced error prone 3’ flap-based Okazaki fragment maturation pathway creates internal tandem duplications for cell survival (RNA-seq)

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Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo

Project description:Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5’ flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2, and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been clearly defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in S. cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5’ flaps, as predicted by some models. Further, using cyclin-controlled constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

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Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

Project description:We investigated the genome-wide distribution of Okazaki fragments in the commonly used laboratory Saccharomyces cerevisiae strain S288C to study the DNA replication model adopted by the budding yeast. The method based upon lambda exonuclease digestion for purification of RNA-primed replication intermediates was first improved to be suitable for the purification of Okazaki fragments. Then, we used this improved method to purify Okazaki fragments from S288C yeast cells, followed by Illumina sequencing. We found that the expected asymmetric distribution of Okazaki fragments around confirmed replication origins, which was derived from the semi-discontinuous DNA replication model, was not observed on S. cerevisiae chromosomes. Even around two highly efficient replication origins, ARS522 and ARS416, the ratios of Okazaki fragments on both strands were inconsistent with the semi-discontinuous DNA replication model. Our study supported the discontinuous DNA replication model. Besides, we also observed that Okazaki fragments were overpresented in the transcribed regions in S. cerevisiae mitochondrial genome, which indicated the interplay between transcription and DNA replication. Examination of the distribution of Okazaki fragments in Saccharomyces cerevisiae strain S288C.

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Next-generation sequencing of Okazaki fragments extracted from Saccharomyces cerevisiae

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Project description:The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.

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