scholarly journals Ku80 removal from DNA through double strand break–induced ubiquitylation

2008 ◽  
Vol 182 (3) ◽  
pp. 467-479 ◽  
Author(s):  
Lisa Postow ◽  
Cristina Ghenoiu ◽  
Eileen M. Woo ◽  
Andrew N. Krutchinsky ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Strand Break ◽  
Double Strand Break ◽  
Nonhomologous End Joining ◽  
Central Component ◽  
End Joining ◽  
Efficient Removal ◽  
Dsb Repair ◽  
Nhej Pathway ◽  
Egg Extract

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1–Cul1–F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.

scholarly journals RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

2005 ◽  
Vol 25 (8) ◽  
pp. 3127-3139 ◽  
Author(s):  
Julie S. Martin ◽  
Nicole Winkelmann ◽  
Mark I. R. Petalcorin ◽  
Michael J. McIlwraith ◽  
Simon J. Boulton
Keyword(s):  
Caenorhabditis Elegans ◽  
Strand Break ◽  
Double Strand Break ◽  
Nonhomologous End Joining ◽  
Related Protein ◽  
End Joining ◽  
Dsb Repair ◽  
Phenotypic Differences ◽  
Dna Double Strand Break ◽  
Radiation Induced

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.

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 Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase

2003 ◽  
Vol 23 (7) ◽  
pp. 2309-2315 ◽  
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.

scholarly journals Nucleosome disassembly during human non-homologous end joining followed by concerted HIRA- and CAF-1-dependent reassembly

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Xuan Li ◽  
Jessica K Tyler
Keyword(s):  
Strand Break ◽  
End Joining ◽  
Efficient Removal ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Nucleosome Disassembly ◽  
End Resection ◽  
Assembly Pathways

The cell achieves DNA double-strand break (DSB) repair in the context of chromatin structure. However, the mechanisms used to expose DSBs to the repair machinery and to restore the chromatin organization after repair remain elusive. Here we show that induction of a DSB in human cells causes local nucleosome disassembly, apparently independently from DNA end resection. This efficient removal of histone H3 from the genome during non-homologous end joining was promoted by both ATM and the ATP-dependent nucleosome remodeler INO80. Chromatin reassembly during DSB repair was dependent on the HIRA histone chaperone that is specific to the replication-independent histone variant H3.3 and on CAF-1 that is specific to the replication-dependent canonical histones H3.1/H3.2. Our data suggest that the epigenetic information is re-established after DSB repair by the concerted and interdependent action of replication-independent and replication-dependent chromatin assembly pathways.

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

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