scholarly journals Resolution of single and double Holliday junction recombination intermediates by GEN1

2017 ◽  
Vol 114 (3) ◽  
pp. 443-450 ◽  
Author(s):  
Rajvee Shah Punatar ◽  
Maria Jose Martin ◽  
Haley D. M. Wyatt ◽  
Ying Wai Chan ◽  
Stephen C. West
Keyword(s):  
Genetic Recombination ◽  
Consensus Sequence ◽  
Holliday Junctions ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Synthetic Dna ◽  
Strict Requirement ◽  
Covalently Linked ◽  
Double Holliday Junction

Genetic recombination provides an important mechanism for the repair of DNA double-strand breaks. Homologous pairing and strand exchange lead to the formation of DNA intermediates, in which sister chromatids or homologous chromosomes are covalently linked by four-way Holliday junctions (HJs). Depending on the type of recombination reaction that takes place, intermediates may have single or double HJs, and their resolution is essential for proper chromosome segregation. In mitotic cells, double HJs are primarily dissolved by the BLM helicase-TopoisomeraseIIIα-RMI1-RMI2 (BTR) complex, whereas single HJs (and double HJs that have escaped the attention of BTR) are resolved by structure-selective endonucleases known as HJ resolvases. These enzymes are ubiquitous in nature, because they are present in bacteriophage, bacteria, archaea, and simple and complex eukaryotes. The human HJ resolvase GEN1 is a member of the XPG/Rad2 family of 5′-flap endonucleases. Biochemical studies of GEN1 revealed that it cleaves synthetic DNA substrates containing a single HJ by a mechanism similar to that shown by the prototypic HJ resolvase,Escherichia coliRuvC protein, but it is unclear whether these substrates fully recapitulate the properties of recombination intermediates that arise within a physiological context. Here, we show that GEN1 efficiently cleaves both single and double HJs contained within large recombination intermediates. Moreover, we find that GEN1 exhibits a weak sequence preference for incision between two G residues that reside in a T-rich region of DNA. These results contrast with those obtained with RuvC, which exhibits a strict requirement for the consensus sequence 5′-A/TTTG/C-3′.

scholarly journals Repeated strand invasion and extensive branch migration are hallmarks of meiotic recombination

2021 ◽  
Author(s):  
Jasvinder S Ahuja ◽  
Catherine S Harvey ◽  
David L Wheeler ◽  
Michael Lichten
Keyword(s):  
Meiotic Recombination ◽  
Holliday Junctions ◽  
Branch Migration ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Strand Invasion ◽  
Break Repair ◽  
Strand Annealing ◽  
Double Holliday Junction ◽  
Dual Mechanism

Currently favored models for meiotic recombination posit that both noncrossover and crossover recombination are initiated by DNA double strand breaks but form by different mechanisms, noncrossovers by synthesis dependent strand annealing, and crossovers by formation and resolution of double Holliday junctions centered around the break. This dual mechanism hypothesis predicts different hybrid DNA patterns in noncrossover and crossover recombinants. We show that these predictions are not upheld, by mapping with unprecedented resolution, parental strand contributions to recombinants at a model locus. Instead, break repair in both noncrossovers and crossovers involves synthesis-dependent strand annealing, often with multiple rounds of strand invasion. Crossover-specific double Holliday junction formation occurs via processes that involve branch migration as an integral feature and that can be separated from break repair itself. These findings reveal meiotic recombination to be a highly dynamic process and prompt a new view of the relationship between crossover and noncrossover recombination.

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

2002 ◽  
Vol 195 (3) ◽  
pp. 309-316 ◽  
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.

scholarly journals Characterization of strand exchange activity of yeast Rad51 protein.

1997 ◽  
Vol 17 (9) ◽  
pp. 5359-5368 ◽  
Author(s):  
E Namsaraev ◽  
P Berg
Keyword(s):  
Genetic Recombination ◽  
Strand Exchange ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Ssdna Binding ◽  
Double Stranded Dna ◽  
Ssdna Binding Protein ◽  
Linear Dsdna ◽  
Circular Ssdna

The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks. Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein. The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide. Linear dsDNAs with recessed complementary or blunt ends are not utilized. The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules. Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.

scholarly journals Concerted cutting by Spo11 illuminates DNA break mechanisms and initiates gap repair during meiosis

2019 ◽  
Author(s):  
Dominic Johnson ◽  
Margaret Crawford ◽  
Tim Cooper ◽  
Corentin Claeys Bouuaert ◽  
Scott Keeney ◽  
...  
Keyword(s):  
Dna Damage Response ◽  
Meiotic Recombination ◽  
Genetic Recombination ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Current Thinking ◽  
Damage Response ◽  
Fixed Orientation ◽  
Dna Break

SummaryGenetic variation arises during meiosis via repair of DNA double-strand breaks (DSBs) created by the topoisomerase-like Spo11 protein. These DSBs are thought to always occur sparsely across the genome, with isolated DSBs generating discrete recombination events. We challenge this view, demonstrating that hyper-localised coincident DSBs frequently form within hotspots in both S. cerevisiae and mouse—a process suppressed by the DNA damage response kinase Tel1/ATM. Remarkably, the distances separating coincident DSBs vary with ∼10.5 bp periodicity, invoking a model where adjacent Spo11 molecules have a fixed orientation relative to the DNA helix. Deep sequencing of meiotic progeny identifies recombination scars consistent with gap repair initiated by adjacent DSBs. Our results revise current thinking about how genetic recombination initiates, reviving original concepts of meiotic recombination as double-strand gap repair.

scholarly journals High-Throughput Screening Approach for Identifying Compounds That Inhibit Nonhomologous End Joining

2017 ◽  
Vol 23 (7) ◽  
pp. 624-633 ◽  
Author(s):  
Andrea L. Bredemeyer ◽  
Bruce S. Edwards ◽  
Mark K. Haynes ◽  
Abigail J. Morales ◽  
Yinan Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cancer Therapeutics ◽  
Inhibitory Effect ◽  
Nonhomologous End Joining ◽  
Recombination Reaction ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Dsbs

DNA double-strand breaks (DSBs) are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Compounds that modulate HR have shown promise as cancer therapeutics. The V(D)J recombination reaction, which assembles antigen receptor genes in lymphocytes, is initiated by the introduction of DNA DSBs at two recombining gene segments by the RAG endonuclease, followed by the NHEJ-mediated repair of these DSBs. Here, using HyperCyt automated flow cytometry, we develop a robust high-throughput screening (HTS) assay for NHEJ that utilizes engineered pre-B-cell lines where the V(D)J recombination reaction can be induced and monitored at a single-cell level. This approach, novel in processing four 384-well plates at a time in parallel, was used to screen the National Cancer Institute NeXT library to identify compounds that inhibit V(D)J recombination and NHEJ. Assessment of cell light scattering characteristics at the primary HTS stage (83,536 compounds) enabled elimination of 60% of apparent hits as false positives. Although all the active compounds that we identified had an inhibitory effect on RAG cleavage, we have established this as an approach that could identify compounds that inhibit RAG cleavage or NHEJ using new chemical libraries.

scholarly journals Removal of Spo11 from meiotic DNA breaksin vitrobut notin vivoby Tyrosyl DNA Phosphodiesterase 2

2019 ◽  
Author(s):  
Dominic Johnson ◽  
Rachal M Allison ◽  
Elda Cannavo ◽  
Petr Cejka ◽  
Matthew J Neale
Keyword(s):  
Meiotic Recombination ◽  
Dna Cleavage ◽  
Protein Complexes ◽  
Dna Lesions ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Alternative Processing ◽  
Covalently Linked

ABSTRACTMeiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes covalently linked to the 5′ ends generated on each side of the DSB. Whilst Spo11-oligos—the product of nucleolytic removal by Mre11—have been detected in a number of biological systems, the lifetime of the covalent Spo11-DSB precursor has not been systematically determined and may be subject to alternative processing reactions. Here we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2, on Spo11-DSBs isolated fromS. cerevisiaecells. We demonstrate that TDP2 can remove Spo11 from natural ssDNA-oligos, and dsDNA ends even when in the presence of excess competitor genomic DNA. Interestingly, TDP2-processed Spo11-DSBs are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essentialin vivoto facilitate resection-dependent HR at Spo11-DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptidesin vitro, TDP2 was unable to remove Spo11in vivo—unlike during the repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher order protein complexes resident at sites of meiotic recombination.

scholarly journals Extensive sex differences at the initiation of genetic recombination

2017 ◽  
Author(s):  
Kevin Brick ◽  
Sarah Thibault-Sennett ◽  
Fatima Smagulova ◽  
Kwan-Wood G. Lam ◽  
Yongmei Pu ◽  
...  
Keyword(s):  
Sex Differences ◽  
Chromosomal Abnormalities ◽  
Genetic Recombination ◽  
Double Strand Breaks ◽  
Local Context ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Genome Wide ◽  
Meiotic Dsbs ◽  
Initiation Stage

AbstractHomologous recombination in meiosis is initiated by programmed DNA double strand breaks (DSBs) and DSB repair as a crossover is essential to prevent chromosomal abnormalities in gametes. Sex differences in recombination have been previously observed by analyses of recombination end-products. To understand when and how sex differences are established, we built genome-wide maps of meiotic DSBs in both male and female mice. We found that most recombination initiates at sex-biased DSB hotspots. Local context, the choice of DSB targeting pathway and sex-specific patterns of DNA methylation give rise to these differences. Sex differences are not limited to the initiation stage, as the rate at which DSBs are repaired as crossovers appears to differ between the sexes in distal regions. This uneven repair patterning may be linked to the higher aneuploidy rate in females. Together, these data demonstrate that sex differences occur early in meiotic recombination.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
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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