After double-strand break induction by UV-A, homologous recombination and nonhomologous end joining cooperate at the same DSB if both systems are available

ABSTRACT The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.

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Radiosensitization by Hyperthermia in the Chicken B-Lymphocyte Cell Line DT40 and its Derivatives Lacking Nonhomologous End Joining and/or Homologous Recombination Pathways of DNA Double-Strand Break Repair

Radiation Research ◽

10.1667/rr3239 ◽

2004 ◽

Vol 162 (4) ◽

pp. 433-441 ◽

Cited By ~ 18

Author(s):

Hong Lan Yin ◽

Yuka Suzuki ◽

Yoshihisa Matsumoto ◽

Masanori Tomita ◽

Yoshiya Furusawa ◽

...

Keyword(s):

Homologous Recombination ◽

Cell Line ◽

B Lymphocyte ◽

Strand Break ◽

Nonhomologous End Joining ◽

Double Strand Break Repair ◽

End Joining ◽

Dna Double Strand Break ◽

Lymphocyte Cell ◽

Lymphocyte Cell Line

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Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase

Molecular and Cellular Biology ◽

10.1128/mcb.23.7.2309-2315.2003 ◽

2003 ◽

Vol 23 (7) ◽

pp. 2309-2315 ◽

Cited By ~ 121

Author(s):

Stephanie A. Nick McElhinny ◽

Dale A. Ramsden

Keyword(s):

High Specificity ◽

Strand Break ◽

Double Strand Break ◽

Nonhomologous End Joining ◽

Double Strand Break Repair ◽

End Joining ◽

Strand Breaks ◽

Break Repair ◽

The Impact

ABSTRACT DNA polymerases are defined as such because they use deoxynucleotides instead of ribonucleotides with high specificity. We show here that polymerase mu (pol μ), implicated in the nonhomologous end-joining pathway for repair of DNA double-strand breaks, incorporates both ribonucleotides and deoxynucleotides in a template-directed manner. pol μ has an approximately 1,000-fold-reduced ability to discriminate against ribonucleotides compared to that of the related pol β, although pol μ's substrate specificity is similar to that of pol β in most other respects. Moreover, pol μ more frequently incorporates ribonucleotides when presented with nucleotide concentrations that approximate cellular pools. We therefore addressed the impact of ribonucleotide incorporation on the activities of factors required for double-strand break repair by nonhomologous end joining. We determined that the ligase required for this pathway readily joined strand breaks with terminal ribonucleotides. Most significantly, pol μ frequently introduced ribonucleotides into the repair junctions of an in vitro nonhomologous end-joining reaction, an activity that would be expected to have important consequences in the context of cellular double-strand break repair.

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

10.1101/2020.05.03.075358 ◽

2020 ◽

Cited By ~ 1

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.

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