Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS. Genome-wide mapping of DNA DSBs in HEK293T cells
Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS.
Project description:Poly [ADP-ribose] polymerase 1 (PARP1) is involved in differentiation, proliferation, tumor transformation, in repair of single-stranded DNA and double-stranded DNA breaks (DSBs) in conjunction with BRCA. PARP1 enzyme works modifying nuclear proteins by poly ADP-ribosylation. It was found that PARP1 and HNRNPA2B1 bind at termini of forum domains and may play a role in coordinated regulation of genes in forum domains (Tchurikov et al., 2013). Forum domains are long DNA fragments of excised chromosomal DNA. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). This ChIP-Seq is aimed to compare genome-wide binding sites of PARP1 with different genomic features, including the hot spots of DSBs.
Project description:4C procedure was used for analysis of genomic contacts of rDNA units in HEK 293T cells. The primers for 4C were selected inside IGS. Our data indicate that mostly rDNA units exhibit close proximity with pericentromeric regions in different chromosomes. We also detected the contacts within a rDNA unit and between rDNA units. Examination of rDNA genome-wide contacts in HEK 293T cells using 4C approach.
Project description:4C procedure was used for analysis of genomic contacts of rDNA units in HEK 293T cells. The primers for 4C were selected inside IGS. Our data indicate that mostly rDNA units exhibit close proximity with pericentromeric regions in different chromosomes. We also detected the contacts within a rDNA unit and between rDNA units.
Project description:Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.
Project description:Colibactin, a potent genotoxin of Escherichia coli, causes DNA double strand breaks (DSBs). We investigated if colibactin creates a particular DNA damage signature in infected human cells. Genomic contexts of colibactin-induced DSBs were enriched for a distinct AT-rich hexameric sequence motif. A survey of somatic mutations at the colibactin target sites of several thousand cancer genomes revealed significant enrichment of the motif in colorectal cancers. Moreover, the exact break point location corresponded with mutational hot spots in these cancers corresponding to a distinct trinucleotide signature. This work provides evidence for a role of colibactin in the etiology of human cancer.
