scholarly journals Comparing Effectiveness of Different Microhomology Lengths in Microhomology Mediated End Joining (MMEJ) Repair of DNA Double Stranded Breaks (DSBs)

2021 ◽  
Vol 9 ◽  
pp. 3-8
Author(s):  
Vasanth Senthilraja ◽  
Rehet Chugh ◽  
Sehej Chugh ◽  
Ethan Yang ◽  
Himanshu Wagh
Keyword(s):  
Repair Process ◽  
Chromosome 15 ◽  
Repair Mechanism ◽  
End Joining ◽  
Base Pairs ◽  
Dsb Repair ◽  
Homologous Chromosomes ◽  
Chemical Agents ◽  
Dna Strands ◽  
His3 Gene

DNA Double-Stranded Breaks (DSBs) are caused by genotoxic agents, such as ionizing radiation and chemical agents, and can cause an affected cell to undergo apoptosis or cell death. The process of microhomology-mediated end joining (MMEJ) shows promising results in the repair of DSBs in DNA. MMEJ is a mutagenic DSB repair mechanism that uses a certain length of homologous nucleotides adjacent to the DSB to align the broken DNA strands for repair. This can result in insertions, deletions, and even translocations of genes at the DSB site. This has led to discussions of debate on whether MMEJ is efficient in repairing DSBs in DNA. Based on the length of microhomology, the effectiveness of the DSB repair can vary. The purpose of this research is to examine MMEJ repair using micro-homologies of different lengths in Saccharomyces cerevisiae cells to test the effectiveness of MMEJ repair. The HIS3 gene located in chromosome 15 in the yeast cell is used to test for MMEJ repair, and the full microhomology length represents 311 base pairs (bp). Various crosses are performed on cells to attain desired genotypes that have the homologous chromosomes in alignment for MMEJ repair. After inducing DSBs, media-based testing is used for testing the efficiency of MMEj repair by checking for the presence of certain genes that may have formed or been deleted during the repair process.

scholarly journals Trapped Topoisomerase II initiates formation of de novo duplications via the nonhomologous end-joining pathway in yeast

2020 ◽  
Author(s):  
Nicole Stantial ◽  
Anna Rogojina ◽  
Matthew Gilbertson ◽  
Yilun Sun ◽  
Hannah Miles ◽  
...  
Keyword(s):  
Topoisomerase Ii ◽  
De Novo ◽  
Strand Break ◽  
Double Strand Break ◽  
Nonhomologous End Joining ◽  
Mutant Form ◽  
Chemotherapeutic Drugs ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Strands

ABSTRACTTopoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5’ end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2- F1025Y,R1128G) whose product generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2- F1025Y,R1128G allele is associated with a novel mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that over-express the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.SIGNIFICANCE STATEMENTDNA-strand separation during transcription and replication creates topological problems that are resolved by topoisomerases. These enzymes nick DNA strands to allow strand passage and then reseal the broken DNA to restore its integrity. Topoisomerase II (Top2) nicks complementary DNA strands to create double-strand break (DSBs) intermediates that can be stabilized by chemotherapeutic drugs and are toxic if not repaired. We identified a mutant form of yeast Top2 that forms stabilized cleavage intermediates in the absence of drugs. Over- expression of the mutant Top2 was associated with a unique mutation signature in which small (1-4 bp), unique segments of DNA were duplicated. These de novo duplications required the nonhomologous end-joining pathway of DSB repair, and their Top2-dependence has clinical and evolutionary implications.

scholarly journals Rapid Recruitment of BRCA1 to DNA Double-Strand Breaks Is Dependent on Its Association with Ku80

2008 ◽  
Vol 28 (24) ◽  
pp. 7380-7393 ◽  
Author(s):  
Leizhen Wei ◽  
Li Lan ◽  
Zehui Hong ◽  
Akira Yasui ◽  
Chikashi Ishioka ◽  
...  
Keyword(s):  
Susceptibility Gene ◽  
Repair Process ◽  
Strand Break ◽  
Missense Mutations ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Nhej Pathway ◽  
C Terminus ◽  
N Terminus

ABSTRACT BRCA1 is the first susceptibility gene to be linked to breast and ovarian cancers. Although mounting evidence has indicated that BRCA1 participates in DNA double-strand break (DSB) repair pathways, its precise mechanism is still unclear. Here, we analyzed the in situ response of BRCA1 at DSBs produced by laser microirradiation. The amino (N)- and carboxyl (C)-terminal fragments of BRCA1 accumulated independently at DSBs with distinct kinetics. The N-terminal BRCA1 fragment accumulated immediately after laser irradiation at DSBs and dissociated rapidly. In contrast, the C-terminal fragment of BRCA1 accumulated more slowly at DSBs but remained at the sites. Interestingly, rapid accumulation of the BRCA1 N terminus, but not the C terminus, at DSBs depended on Ku80, which functions in the nonhomologous end-joining (NHEJ) pathway, independently of BARD1, which binds to the N terminus of BRCA1. Two small regions in the N terminus of BRCA1 independently accumulated at DSBs and interacted with Ku80. Missense mutations found within the N terminus of BRCA1 in cancers significantly changed the kinetics of its accumulation at DSBs. A P142H mutant failed to associate with Ku80 and restore resistance to irradiation in BRCA1-deficient cells. These might provide a molecular basis of the involvement of BRCA1 in the NHEJ pathway of the DSB repair process.

scholarly journals Gene conversion adjacent to regions of double-strand break repair.

1988 ◽  
Vol 8 (12) ◽  
pp. 5292-5298 ◽  
Author(s):  
T L Orr-Weaver ◽  
A Nicolas ◽  
J W Szostak
Keyword(s):  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Yeast Cells ◽  
Double Strand Break Repair ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Break Repair ◽  
His3 Gene

The repair of double-strand breaks and gaps can be studied in vegetative yeast cells by transforming the DNA with restriction enzyme-cut plasmids. Postulated models for this repair process require the formation of heteroduplex DNA on either side of the region of break or gap repair. We describe the use of restriction site mutations in the his3 gene to detect conversion events flanking but outside of a region of a double-strand break repair. The frequency with which a mutation was converted declined with increasing distance between the mutation and the edge of the gap repair region. The data are consistent with heteroduplex DNA tracts of at least several hundred base pairs adjacent to regions of double-strand break repair.

scholarly journals A pathway for error-free non-homologous end joining of resected meiotic double-strand breaks

2020 ◽  
Author(s):  
Talia Hatkevich ◽  
Danny E. Miller ◽  
Carolyn A. Turcotte ◽  
Margaret C. Miller ◽  
Jeff Sekelsky
Keyword(s):  
Meiotic Recombination ◽  
Homologous Chromosome ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Repair Mechanism ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Recombination Processes ◽  
Non Homologous End Joining

ABSTRACTProgrammed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic divisions. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. We describe a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. Our results provide support for a recombination intermediate recently discovered in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. We propose that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ.

Gene conversion adjacent to regions of double-strand break repair

1988 ◽  
Vol 8 (12) ◽  
pp. 5292-5298
Author(s):  
T L Orr-Weaver ◽  
A Nicolas ◽  
J W Szostak
Keyword(s):  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Yeast Cells ◽  
Double Strand Break Repair ◽  
Base Pairs ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Break Repair ◽  
His3 Gene

The repair of double-strand breaks and gaps can be studied in vegetative yeast cells by transforming the DNA with restriction enzyme-cut plasmids. Postulated models for this repair process require the formation of heteroduplex DNA on either side of the region of break or gap repair. We describe the use of restriction site mutations in the his3 gene to detect conversion events flanking but outside of a region of a double-strand break repair. The frequency with which a mutation was converted declined with increasing distance between the mutation and the edge of the gap repair region. The data are consistent with heteroduplex DNA tracts of at least several hundred base pairs adjacent to regions of double-strand break repair.

scholarly journals The Chromatin Response to Double-Strand DNA Breaks and Their Repair

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1853
Author(s):  
Radoslav Aleksandrov ◽  
Rossitsa Hristova ◽  
Stoyno Stoynov ◽  
Anastas Gospodinov
Keyword(s):  
Dna Repair ◽  
Cancer Progression ◽  
Repair Process ◽  
Dna Breaks ◽  
Dsb Repair ◽  
Double Strand Dna Breaks ◽  
Dna Repair Proteins ◽  
Dna Strands ◽  
Cellular Dna ◽  
Double Strand Dna

Cellular DNA is constantly being damaged by numerous internal and external mutagenic factors. Probably the most severe type of insults DNA could suffer are the double-strand DNA breaks (DSBs). They sever both DNA strands and compromise genomic stability, causing deleterious chromosomal aberrations that are implicated in numerous maladies, including cancer. Not surprisingly, cells have evolved several DSB repair pathways encompassing hundreds of different DNA repair proteins to cope with this challenge. In eukaryotic cells, DSB repair is fulfilled in the immensely complex environment of the chromatin. The chromatin is not just a passive background that accommodates the multitude of DNA repair proteins, but it is a highly dynamic and active participant in the repair process. Chromatin alterations, such as changing patterns of histone modifications shaped by numerous histone-modifying enzymes and chromatin remodeling, are pivotal for proficient DSB repair. Dynamic chromatin changes ensure accessibility to the damaged region, recruit DNA repair proteins, and regulate their association and activity, contributing to DSB repair pathway choice and coordination. Given the paramount importance of DSB repair in tumorigenesis and cancer progression, DSB repair has turned into an attractive target for the development of novel anticancer therapies, some of which have already entered the clinic.

scholarly journals HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

Nature ◽  
2021 ◽  
Author(s):  
Roopesh Anand ◽  
Erika Buechelmaier ◽  
Ondrej Belan ◽  
Matthew Newton ◽  
Aleksandra Vancevska ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cellular Level ◽  
Dual Function ◽  
Dna Unwinding ◽  
End Joining ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
Dna Strands ◽  
Strand Annealing ◽  
Dna Strand

AbstractDNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development1. HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours2–4. At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage3,5. Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear3,5,6. Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51. Using biochemistry analyses and single-molecule imaging, we establish that RAD51 forms a complex with and strongly stimulates HELQ as it translocates during DNA unwinding. By contrast, RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing. Mechanistically, we show that HELQ possesses an intrinsic ability to capture RPA-bound DNA strands and then displace RPA to facilitate annealing of complementary sequences. Finally, we show that HELQ deficiency in cells compromises single-strand annealing and microhomology-mediated end-joining pathways and leads to bias towards long-tract gene conversion tracts during homologous recombination. Thus, our results implicate HELQ in multiple arms of DSB repair through co-factor-dependent modulation of intrinsic translocase and DNA strand annealing activities.

scholarly journals Multiple Pathways for Repair of DNA Double-Strand Breaks in Mammalian Chromosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8353-8360 ◽  
Author(s):  
Yunfu Lin ◽  
Tamas Lukacsovich ◽  
Alan S. Waldman
Keyword(s):  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Base Pairs ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Strand Annealing

ABSTRACT To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.

scholarly journals Quantitative analysis shows that repair of Cas9-induced double-strand DNA breaks is slow and error-prone

2017 ◽  
Author(s):  
Eva K. Brinkman ◽  
Tao Chen ◽  
Marcel de Haas ◽  
Hanna A. Holland ◽  
Waseem Akhtar ◽  
...  
Keyword(s):  
Repair Process ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Double Strand Dna Breaks ◽  
Chemical Inhibition ◽  
Changes Over Time ◽  
Kinetics Of

SummaryThe RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.

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