scholarly journals RSC Mobilizes Nucleosomes To Improve Accessibility of Repair Machinery to the Damaged Chromatin

2006 ◽  
Vol 27 (5) ◽  
pp. 1602-1613 ◽  
Author(s):  
Eun Yong Shim ◽  
Soo Jin Hong ◽  
Ji-Hyun Oum ◽  
Yvonne Yanez ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Base Pairs ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Free Dna ◽  
Key Questions

ABSTRACT Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5′-to-3′ degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.

scholarly journals The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1430
Author(s):  
Ian Yannuzzi ◽  
Margaret A. Butler ◽  
Joel Fernandez ◽  
Jeannine R. LaRocque
Keyword(s):  
Direct Repeat ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna End Resection ◽  
The Impact ◽  
End Resection

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, we affirmed the importance of end resection on DSB repair pathway choice in multicellular systems, described the function of DmCtIP in short and extensive DNA end resection, and determined the impact of end resection on GCT length during HR.

scholarly journals Genome integrity and neurogenesis of postnatal hippocampal neural stem/progenitor cells require a unique regulator Filia

2020 ◽  
Vol 6 (44) ◽  
pp. eaba0682 ◽  
Author(s):  
Jingzheng Li ◽  
Yafang Shang ◽  
Lin Wang ◽  
Bo Zhao ◽  
Chunli Sun ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Neurodevelopmental Disorders ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Replication Forks ◽  
Proof Of Concept ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Neural Stem Progenitor Cells

Endogenous DNA double-strand breaks (DSBs) formation and repair in neural stem/progenitor cells (NSPCs) play fundamental roles in neurogenesis and neurodevelopmental disorders. NSPCs exhibit heterogeneity in terms of lineage fates and neurogenesis activity. Whether NSPCs also have heterogeneous regulations on DSB formation and repair to accommodate region-specific neurogenesis has not been explored. Here, we identified a regional regulator Filia, which is predominantly expressed in mouse hippocampal NSPCs after birth and regulates DNA DSB formation and repair. On one hand, Filia protects stalling replication forks and prevents the replication stress-associated DNA DSB formation. On the other hand, Filia facilitates the homologous recombination–mediated DNA DSB repair. Consequently, Filia−/− mice had impaired hippocampal NSPC proliferation and neurogenesis and were deficient in learning, memory, and mood regulations. Thus, our study provided the first proof of concept demonstrating the region-specific regulations of DSB formation and repair in subtypes of NSPCs.

scholarly journals Human RNF169 is a negative regulator of the ubiquitin-dependent response to DNA double-strand breaks

2012 ◽  
Vol 197 (2) ◽  
pp. 189-199 ◽  
Author(s):  
Maria Poulsen ◽  
Claudia Lukas ◽  
Jiri Lukas ◽  
Simon Bekker-Jensen ◽  
Niels Mailand
Keyword(s):  
Deoxyribonucleic Acid ◽  
Double Strand Breaks ◽  
Negative Regulator ◽  
Nonhomologous End Joining ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Protein Recruitment

Nonproteolytic ubiquitylation of chromatin surrounding deoxyribonucleic acid double-strand breaks (DSBs), mediated by the RNF8/RNF168 ubiquitin ligases, plays a key role in recruiting repair factors, including 53BP1 and BRCA1, to reestablish genome integrity. In this paper, we show that human RNF169, an uncharacterized E3 ubiquitin ligase paralogous to RNF168, accumulated in DSB repair foci through recognition of RNF168-catalyzed ubiquitylation products by its motif interacting with ubiquitin domain. Unexpectedly, RNF169 was dispensable for chromatin ubiquitylation and ubiquitin-dependent accumulation of repair factors at DSB sites. Instead, RNF169 functionally competed with 53BP1 and RAP80–BRCA1 for association with RNF168-modified chromatin independent of its catalytic activity, limiting the magnitude of their recruitment to DSB sites. By delaying accumulation of 53BP1 and RAP80 at damaged chromatin, RNF169 stimulated homologous recombination and restrained nonhomologous end joining, affecting cell survival after DSB infliction. Our results show that RNF169 functions in a noncanonical fashion to harness RNF168-mediated protein recruitment to DSB-containing chromatin, thereby contributing to regulation of DSB repair pathway utilization.

scholarly journals The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009807
Author(s):  
Marco Gnugnoli ◽  
Erika Casari ◽  
Maria Pia Longhese
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atpase Activity ◽  
Long Range ◽  
Chromatin Remodeling ◽  
Short Range ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Strands

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.

scholarly journals The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

2012 ◽  
Vol 199 (7) ◽  
pp. 1067-1081 ◽  
Author(s):  
Céline Courilleau ◽  
Catherine Chailleux ◽  
Alain Jauneau ◽  
Fanny Grimal ◽  
Sébastien Briois ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Remodeling ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna Damage Signaling ◽  
Dna Dsbs ◽  
Dependent Processes

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate–dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.

scholarly journals Coupling crossover and synaptonemal complex in meiosis

2022 ◽  
Vol 36 (1-2) ◽  
pp. 4-6
Author(s):  
Corinne Grey ◽  
Bernard de Massy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Synaptonemal Complex ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Homologous Chromosomes

During meiosis, a molecular program induces DNA double-strand breaks (DSBs) and their repair by homologous recombination. DSBs can be repaired with or without crossovers. ZMM proteins promote the repair toward crossover. The sites of DSB repair are also sites where the axes of homologous chromosomes are juxtaposed and stabilized, and where a structure called the synaptonemal complex initiates, providing further regulation of both DSB formation and repair. How crossover formation and synapsis initiation are linked has remained unknown. The study by Pyatnitskaya and colleagues (pp. 53–69) in this issue of Genes & Development highlights the central role of the Saccharomyces cerevisiae ZMM protein Zip4 in this process.

scholarly journals DNA Double-Strand Break Repair: All Roads Lead to HeterochROMAtin Marks

2021 ◽  
Vol 12 ◽  
Author(s):  
Pierre Caron ◽  
Enrico Pobega ◽  
Sophie E. Polo
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
The Impact

In response to DNA double-strand breaks (DSBs), chromatin modifications orchestrate DNA repair pathways thus safeguarding genome integrity. Recent studies have uncovered a key role for heterochromatin marks and associated factors in shaping DSB repair within the nucleus. In this review, we present our current knowledge of the interplay between heterochromatin marks and DSB repair. We discuss the impact of heterochromatin features, either pre-existing in heterochromatin domains or de novo established in euchromatin, on DSB repair pathway choice. We emphasize how heterochromatin decompaction and mobility further support DSB repair, focusing on recent mechanistic insights into these processes. Finally, we speculate about potential molecular players involved in the maintenance or the erasure of heterochromatin marks following DSB repair, and their implications for restoring epigenome function and integrity.

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 Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

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