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 MSH2-MSH3 promotes DNA end resection during HR and blocks TMEJ through interaction with SMARCAD1 and EXO1

2021 ◽  
Author(s):  
Jung-Min Oh ◽  
Yujin Kang ◽  
Jumi Park ◽  
Yubin Sung ◽  
Dayoung Kim ◽  
...  
Keyword(s):  
Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Direct Role ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
Template Dna ◽  
End Resection

SUMMARYDNA double strand break (DSB) repair by Homologous recombination (HR) is initiated by the end resection, a process during which 3’ ssDNA overhangs are generated by the nucleolytic degradation. The extent of DNA end resection determines the choice of the DSB repair pathway. The role of several proteins including nucleases for end resection has been studied in detail. However, it is still unclear how the initial, nicked DNA generated by MRE11-RAD50-NBS1 is recognized and how subsequent proteins including EXO1 are recruited to DSB sites to facilitate extensive end resection. We found that the MutSβ (MSH2-MSH3) mismatch repair (MMR) complex is recruited to DSB sites by recognizing the initial nicked DNA at DSB sites through the interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 at DSB sites helps to recruit EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 furthermore inhibits the access of DNA polymerase θ (POLQ), which promotes polymerase theta-mediated end-joining (TMEJ) of DSB. Collectively, our data show a direct role for MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing DSB repair pathway by favoring HR over TMEJ. Our findings extend the importance of MMR in DSB repair beyond established role in rejecting the invasion of sequences not perfectly homologous to template DNA during late-stage HR.

scholarly journals MicroRNAs down-regulate homologous recombination in the G1 phase of cycling cells to maintain genomic stability

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Young Eun Choi ◽  
Yunfeng Pan ◽  
Eunmi Park ◽  
Panagiotis Konstantinopoulos ◽  
Subhajyoti De ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
G1 Phase ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Brca1 Brca2 ◽  
G1 Cells

Homologous recombination (HR)-mediated repair of DNA double-strand break (DSB)s is restricted to the post-replicative phases of the cell cycle. Initiation of HR in the G1 phase blocks non-homologous end joining (NHEJ) impairing DSB repair. Completion of HR in G1 cells can lead to the loss-of-heterozygosity (LOH), which is potentially carcinogenic. We conducted a gain-of-function screen to identify miRNAs that regulate HR-mediated DSB repair, and of these miRNAs, miR-1255b, miR-148b*, and miR-193b* specifically suppress the HR-pathway in the G1 phase. These miRNAs target the transcripts of HR factors, BRCA1, BRCA2, and RAD51, and inhibiting miR-1255b, miR-148b*, and miR-193b* increases expression of BRCA1/BRCA2/RAD51 specifically in the G1-phase leading to impaired DSB repair. Depletion of CtIP, a BRCA1-associated DNA end resection protein, rescues this phenotype. Furthermore, deletion of miR-1255b, miR-148b*, and miR-193b* in independent cohorts of ovarian tumors correlates with significant increase in LOH events/chromosomal aberrations and BRCA1 expression.

scholarly journals Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation

Science ◽  
2010 ◽  
Vol 329 (5997) ◽  
pp. 1348-1353 ◽  
Author(s):  
Abderrahmane Kaidi ◽  
Brian T. Weinert ◽  
Chunaram Choudhary ◽  
Stephen P. Jackson
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Protein A ◽  
Strand Break ◽  
Dsb Repair ◽  
Interacting Protein ◽  
Dna Double Strand Break ◽  
Dna End Resection ◽  
Lysine Deacetylases ◽  
End Resection

SIRT6 belongs to the sirtuin family of protein lysine deacetylases, which regulate aging and genome stability. We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accumulation of replication protein A and single-stranded DNA at DNA damage sites, reduced rates of homologous recombination, and sensitized cells to DSB-inducing agents. We identified the DSB resection protein CtIP [C-terminal binding protein (CtBP) interacting protein] as a SIRT6 interaction partner and showed that SIRT6-dependent CtIP deacetylation promotes resection. A nonacetylatable CtIP mutant alleviated the effect of SIRT6 depletion on resection, thus identifying CtIP as a key substrate by which SIRT6 facilitates DSB processing and homologous recombination. These findings further clarify how SIRT6 promotes genome stability.

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.

scholarly journals DNA end-resection in highly accessible chromatin produces a toxic break

2019 ◽  
Author(s):  
Jeroen van den Berg ◽  
Stacey E.P. Joosten ◽  
YongSoo Kim ◽  
Anna G. Manjón ◽  
Lenno Krenning ◽  
...  
Keyword(s):  
Strand Break ◽  
Open Chromatin ◽  
End Joining ◽  
Close Proximity ◽  
Damage Response ◽  
Hypersensitive Sites ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
End Resection ◽  
Accessible Chromatin

AbstractOf all damage occurring to DNA, the double strand break (DSB) is the most toxic lesion. Luckily, cells have developed multiple repair pathways to cope with these lesions. These different pathways compete for the same break, and the location of the break can influence this competition. However, the exact contribution of break location in repair pathway preference is not fully understood. We observe that most breaks prefer classical non-homologous end-joining, whereas some depend on DNA end-resection for their repair. Surprisingly, we find that for a subset of these sites, the activation of resection-dependent repair induces a detrimental DNA damage response. These sites exhibit extensive DNA end-resection due to improper recruitment of 53BP1 and the Shieldin complex due to low levels of H4K20me2. Most of these sites reside in close proximity to DNAseI hypersensitive sites. Compacting or removing these regions reduces extensive DNA end-resection and restores normal repair. Taken together, we found that DSB in open chromatin is highly toxic, due to the improper activity of 53BP1 and Shieldin, resulting in extensive DNA end-resection.

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

2020 ◽  
Author(s):  
Ruben Schep ◽  
Eva K. Brinkman ◽  
Christ Leemans ◽  
Xabier Vergara ◽  
Ben Morris ◽  
...  
Keyword(s):  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

scholarly journals Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

2022 ◽  
Author(s):  
Aditya Mojumdar ◽  
Nancy Adam ◽  
Jennifer A Cobb
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Repair Pathway ◽  
End Joining ◽  
C Terminus ◽  
Dna Double Strand Break ◽  
Canonical Pathways ◽  
Pathway Choice ◽  
Non Homologous End Joining ◽  
End Resection

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330- 338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection.

scholarly journals Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks

2021 ◽  
Author(s):  
Aditya Mojumdar ◽  
Nancy Adam ◽  
Jennifer A Cobb
Keyword(s):  
Synthetic Lethality ◽  
Nuclease Activity ◽  
Strand Break ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Genomic Deletions ◽  
Dna Double Strand Break ◽  
Break Site ◽  
Dna End Resection ◽  
Non Homologous End Joining

The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. Regulated 5 DNA end resection is important for repair pathway choice and repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and, more recently, it was shown to be important for DNA end-bridging and inhibiting Dna2-Sgs1 mediated 5 resection. Dna2 localizes to DSBs in the absence of Sgs1 through interactions with Mre11 and Sae2 and DNA damage sensitivity is greater in cells lacking Dna2 nuclease activity compared to sgs1∆ mutants. Dna2-Sae2 mediated 5 resection is down-regulated by Nej1, which itself interacts with Sae2. The resection defect of sae2∆ and the synthetic lethality of sae2∆ sgs1∆ are reversed by deletion of NEJ1 and dependent on Dna2 nuclease activity. Our work demonstrates the importance of Nej1 in inhibiting short-range resection at a DSB by Dna2-Sae2, a critical regulatory mechanism that prevents the formation of genomic deletions at the break site.

scholarly journals A genetically encoded inhibitor of 53BP1 to stimulate homology-based gene editing

2016 ◽  
Author(s):  
Marella D. Canny ◽  
Leo C.K. Wan ◽  
Amélie Fradet-Turcotte ◽  
Alexandre Orthwein ◽  
Nathalie Moatti ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Cell Types ◽  
Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Non Homologous End Joining ◽  
G1 Cells ◽  
Programmable Nucleases ◽  
End Resection

AbstractThe expanding repertoire of programmable nucleases such as Cas9 brings new opportunities in genetic medicine1–3. In many cases, these nucleases are engineered to induce a DNA double-strand break (DSB) to stimulate precise genome editing by homologous recombination (HR). However, HR efficiency is nearly always hindered by competing DSB repair pathways such as non-homologous end-joining (NHEJ). HR is also profoundly suppressed in non-replicating cells, thus precluding the use of homology-based genome engineering in a wide variety4 of cell types. Here, we report the development of a genetically encoded inhibitor of 53BP1 (known as TP53BP1), a regulator of DSB repair pathway choice5. 53BP1 promotes NHEJ over HR by suppressing end resection, the formation of 3-prime single-stranded DNA tails, which is the rate-limiting step in HR initiation. 53BP1 also blocks the recruitment of the HR factor BRCA1 to DSB sites in G1 cells4,6. The inhibitor of 53BP1 (or i53) was identified through the screening of a massive combinatorial library of engineered ubiquitin variants by phage display7. i53 binds and occludes the ligand binding site of the 53BP1 Tudor domain with high affinity and selectivity, blocking its ability to accumulate at sites of DNA damage. i53 is a potent selective inhibitor of 53BP1 and enhances gene targeting and chromosomal gene conversion, two HR-dependent reactions. Finally, i53 can also activate HR in G1 cells when combined with the activation of end-resection and KEAP1 inhibition. We conclude that 53BP1 inhibition is a robust tool to enhance precise genome editing by canonical HR pathways.

Targeting DNA Double-Strand Break (DSB) Repair to Counteract Tumor Radio-resistance

2019 ◽  
Vol 20 (9) ◽  
pp. 891-902 ◽  
Author(s):  
Yucui Zhao ◽  
Siyu Chen
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Cancer Therapies ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Damages ◽  
Dna Double Strand Break ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Made In

During the last decade, advances of radiotherapy (RT) have been made in the clinical practice of cancer treatment. RT exerts its anticancer effect mainly via leading to the DNA Double-Strand Break (DSB), which is one of the most toxic DNA damages. Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) are two major DSB repair pathways in human cells. It is known that dysregulations of DSB repair elicit a predisposition to cancer and probably result in resistance to cancer therapies including RT. Therefore, targeting the DSB repair presents an attractive strategy to counteract radio-resistance. In this review, we describe the latest knowledge of the two DSB repair pathways, focusing on several key proteins contributing to the repair, such as DNA-PKcs, RAD51, MRN and PARP1. Most importantly, we discuss the possibility of overcoming radiation resistance by targeting these proteins for therapeutic inhibition. Recent tests of DSB repair inhibitors in the laboratory and their translations into clinical studies are also addressed.

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