A novel quantitative electrochemical method to monitor DNA double-strand breaks caused by a DNA cleavage agent at a DNA sensor

Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. In male and female meiotic cells, DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identified their conserved interacting domains by structural analysis. We then analysed the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity was strongly reduced genome-wide in oocytes, but only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity was delayed in autosomes. These results provide evidence that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity by integrating specific chromosomal features.

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Restrictions Limiting the Generation of DNA Double Strand Breaks during Chromosomal V(D)J Recombination

Journal of Experimental Medicine ◽

10.1084/jem.20011803 ◽

2002 ◽

Vol 195 (3) ◽

pp. 309-316 ◽

Cited By ~ 16

Author(s):

Robert E. Tillman ◽

Andrea L. Wooley ◽

Maureen M. Hughes ◽

Tara D. Wehrly ◽

Wojciech Swat ◽

...

Keyword(s):

Dna Cleavage ◽

Receptor Gene ◽

Antigen Receptor ◽

Double Strand Breaks ◽

Recombination Reaction ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Recombination Signal Sequences ◽

Dna Dsbs ◽

Cleavage Step

Antigen receptor loci are composed of numerous variable (V), diversity (D), and joining (J) gene segments, each flanked by recombination signal sequences (RSSs). The V(D)J recombination reaction proceeds through RSS recognition and DNA cleavage steps making it possible for multiple DNA double strand breaks (DSBs) to be introduced at a single locus. Here we use ligation-mediated PCR to analyze DNA cleavage intermediates in thymocytes from mice with targeted RSS mutations at the endogenous TCRβ locus. We show that DNA cleavage does not occur at individual RSSs but rather must be coordinated between RSS pairs flanking gene segments that ultimately form coding joins. Coordination of the DNA cleavage step occurs over great distances in the chromosome and favors intra- over interchromosomal recombination. Furthermore, through several restrictions imposed on the generation of both nonpaired and paired DNA DSBs, this requirement promotes antigen receptor gene integrity and genomic stability in developing lymphocytes undergoing V(D)J recombination.

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Emodin Triggers DNA Double-Strand Breaks by Stabilizing Topoisomerase II-DNA Cleavage Complexes and by Inhibiting ATP Hydrolysis of Topoisomerase II

Toxicological Sciences ◽

10.1093/toxsci/kfq282 ◽

2010 ◽

Vol 118 (2) ◽

pp. 435-443 ◽

Cited By ~ 40

Author(s):

Yan Li ◽

Yang Luan ◽

Xinming Qi ◽

Ming Li ◽

Likun Gong ◽

...

Keyword(s):

Dna Cleavage ◽

Topoisomerase Ii ◽

Atp Hydrolysis ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Hydrolysis Of

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Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands

Chemical Science ◽

10.1039/c6sc04094b ◽

2017 ◽

Vol 8 (5) ◽

pp. 3726-3740 ◽

Cited By ~ 21

Author(s):

Cynthia Griffith ◽

Adam S. Dayoub ◽

Thamara Jaranatne ◽

Nagham Alatrash ◽

Ali Mohamedi ◽

...

Keyword(s):

Dna Cleavage ◽

Double Strand Breaks ◽

Time Dependent ◽

Dna Double Strand Breaks ◽

Ruthenium Polypyridyl ◽

Polypyridyl Complexes ◽

Strand Breaks ◽

Redox Active ◽

Ruthenium Polypyridyl Complexes ◽

Catalytic Dna

Yellow foci show time dependent DNA double strand breaks in the nuclei of H358 cells treated with IC50 concentration of [(phen)2Ru(tatpp)Ru(Phen)2]Cl4.

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CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

10.1101/2021.09.08.459484 ◽

2021 ◽

Author(s):

Jun Huang ◽

David Rowe ◽

Wei Zhang ◽

Tyler Suelter ◽

Barbara Valent ◽

...

Keyword(s):

Dna Repair ◽

Dna Cleavage ◽

Large Scale ◽

Genome Engineering ◽

Natural Populations ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Long Read ◽

Repair Pathways

AbstractCRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.

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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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