Single nucleotide-level mapping of DNA double-strand breaks in human HEK293T cells

Identification of a signature of evolutionarily conserved stress-induced mutagenesis in cancer

10.1101/2021.04.17.440291 ◽

2021 ◽

Author(s):

Luis Humberto Cisneros ◽

Kimberly J Bussey ◽

Charles Vasque

Keyword(s):

Double Strand Breaks ◽

Whole Genome Sequencing Data ◽

Dna Double Strand Breaks ◽

Sequencing Data ◽

Single Nucleotide ◽

Strand Breaks ◽

Evolutionarily Conserved ◽

Induced Mutagenesis ◽

Genomic Locations ◽

Inherited Mutations

The clustering of mutations observed in cancer cells is reminiscent of the stress-induced mutagenesis (SIM) response in bacteria. SIM employs error-prone polymerases resulting in mutations concentrated around DNA double-strand breaks with an abundance that decays with genomic distance. We performed a quantitative study on single nucleotide variant calls for whole-genome sequencing data from 1950 tumors and non-inherited mutations from 129 normal samples. We introduce statistical methods to identify mutational clusters and quantify their distribution pattern. Our results show that mutations in both normal and cancer samples are indeed clustered and have shapes indicative of SIM. We found the genomic locations of groups of close mutations are more likely to be prevalent across normal samples than in cancer suggesting loss of regulation over the mutational process during carcinogenesis.

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Locally, Meiotic Double-Strand Breaks Targeted by Gal4BD-Spo11 Occur at Discrete Sites with a Sequence Preference

Molecular and Cellular Biology ◽

10.1128/mcb.00088-09 ◽

2009 ◽

Vol 29 (13) ◽

pp. 3500-3516 ◽

Cited By ~ 19

Author(s):

Hajime Murakami ◽

Alain Nicolas

Keyword(s):

Hot Spots ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Single Nucleotide ◽

Strand Breaks ◽

Sequence Preference ◽

Meiotic Dsbs ◽

Resolution Mapping ◽

Nucleotide Resolution ◽

Recombination Hot Spots

ABSTRACT Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within ∼75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a “DSB targeting window.” Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.

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CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

10.1101/582403 ◽

2019 ◽

Cited By ~ 2

Author(s):

Yaojun Tong ◽

Helene L. Robertsen ◽

Kai Blin ◽

Andreas K. Klitgaard ◽

Tilmann Weber ◽

...

Keyword(s):

Genome Instability ◽

Cytidine Deaminase ◽

Double Strand Breaks ◽

Antimicrobial Compounds ◽

Dna Double Strand Breaks ◽

Single Nucleotide ◽

Strand Breaks ◽

Genetic Manipulations ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractFilamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

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Repair pathway choice for double-strand breaks

Essays in Biochemistry ◽

10.1042/ebc20200007 ◽

2020 ◽

Vol 64 (5) ◽

pp. 765-777 ◽

Cited By ~ 2

Author(s):

Yixi Xu ◽

Dongyi Xu

Keyword(s):

Cell Cycle ◽

Double Strand Breaks ◽

Homologous Region ◽

Gene Repair ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

Environmental Radiation ◽

Strand Breaks ◽

Dna End Resection ◽

End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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