The human HELLS chromatin remodelling protein promotes end resection to facilitate homologous recombination within heterochromatin

2018 ◽  
Author(s):  
G. Kollarovic ◽  
C. E. Topping ◽  
E. P. Shaw ◽  
A. L. Chambers
Keyword(s):  
Homologous Recombination ◽  
Cancer Biology ◽  
Genome Stability ◽  
Strand Break ◽  
Chromatin Remodelling ◽  
Dsb Repair ◽  
Icf Syndrome ◽  
Damage Response ◽  
Break Repair ◽  
End Resection

ABSTRACTEfficient double-strand break repair in eukaryotes requires manipulation of chromatin structure. ATP-dependent chromatin remodelling enzymes can facilitate different DNA repair pathways, during different stages of the cell cycle and in a range of chromatin environments. The contribution of remodelling factors to break repair within heterochromatin during G2 is unclear.The human HELLS protein is a Snf2-like chromatin remodeller family member and is mutated or misregulated in several cancers and some cases of ICF syndrome. HELLS has been implicated in the DNA damage response, but its mechanistic function in repair is not well understood. We find that HELLS facilitates homologous recombination at two-ended breaks within heterochromatic regions during G2. HELLS enables end-resection and accumulation of CtIP at IR-induced foci. We identify an interaction between HELLS and CtIP and establish that the ATPase domain of HELLS is required to promote DSB repair. This function of HELLS in maintenance of genome stability is likely to contribute to its role in cancer biology and demonstrates that different chromatin remodelling activities are required for efficient repair in specific genomic contexts.

scholarly journals The human HELLS chromatin remodelling protein promotes end resection to facilitate homologous recombination and contributes to DSB repair within heterochromatin

2019 ◽  
Vol 48 (4) ◽  
pp. 1872-1885 ◽  
Author(s):  
Gabriel Kollárovič ◽  
Caitríona E Topping ◽  
Edward P Shaw ◽  
Anna L Chambers
Keyword(s):  
Homologous Recombination ◽  
Cancer Biology ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Break Repair ◽  
End Resection

Abstract Efficient double-strand break repair in eukaryotes requires manipulation of chromatin structure. ATP-dependent chromatin remodelling enzymes facilitate different DNA repair pathways, during different stages of the cell cycle and in varied chromatin environments. The contribution of remodelling factors to double-strand break repair within heterochromatin during G2 is unclear. The human HELLS protein is a Snf2-like chromatin remodeller family member and is mutated or misregulated in several cancers and some cases of ICF syndrome. HELLS has been implicated in the DNA damage response, but its mechanistic function in repair is not well understood. We discover that HELLS facilitates homologous recombination at two-ended breaks and contributes to repair within heterochromatic regions during G2. HELLS promotes initiation of HR by facilitating end-resection and accumulation of CtIP at IR-induced foci. We identify an interaction between HELLS and CtIP and establish that the ATPase domain of HELLS is required to promote DSB repair. This function of HELLS in maintenance of genome stability is likely to contribute to its role in cancer biology and demonstrates that different chromatin remodelling activities are required for efficient repair in specific genomic contexts.

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 RAD6 Promotes Homologous Recombination Repair by Activating the Autophagy-Mediated Degradation of Heterochromatin Protein HP1

2014 ◽  
Vol 35 (2) ◽  
pp. 406-416 ◽  
Author(s):  
Su Chen ◽  
Chen Wang ◽  
Luxi Sun ◽  
Da-Liang Wang ◽  
Lu Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Strand Break ◽  
Open Chromatin ◽  
Dsb Repair ◽  
Heterochromatin Protein ◽  
Dna Double Strand Break ◽  
Recombination Repair ◽  
Ubiquitin Conjugating Enzyme

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genome stability. Unrepaired or misrepaired DSBs cause chromosomal rearrangements that can result in severe consequences, such as tumorigenesis. RAD6 is an E2 ubiquitin-conjugating enzyme that plays a pivotal role in repairing UV-induced DNA damage. Here, we present evidence that RAD6 is also required for DNA DSB repair via homologous recombination (HR) by specifically regulating the degradation of heterochromatin protein 1α (HP1α). Our study indicates that RAD6 physically interacts with HP1α and ubiquitinates HP1α at residue K154, thereby promoting HP1α degradation through the autophagy pathway and eventually leading to an open chromatin structure that facilitates efficient HR DSB repair. Furthermore, bioinformatics studies have indicated that the expression of RAD6 and HP1α exhibits an inverse relationship and correlates with the survival rate of patients.

scholarly journals Tetratricopeptide repeat factor XAB2 mediates the end resection step of homologous recombination

2016 ◽  
Vol 44 (12) ◽  
pp. 5702-5716 ◽  
Author(s):  
David O Onyango ◽  
Sean M Howard ◽  
Kashfia Neherin ◽  
Diana A Yanez ◽  
Jeremy M Stark
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Dna Damage Response ◽  
Strand Break ◽  
Tetratricopeptide Repeat ◽  
Intermediate Step ◽  
Damage Response ◽  
Interchromatin Granules ◽  
Chromosomal Break ◽  
End Resection

Abstract We examined the influence of the tetratricopeptide repeat factor XAB2 on chromosomal break repair, and found that XAB2 promotes end resection that generates the 3′ ssDNA intermediate for homologous recombination (HR). Namely, XAB2 is important for chromosomal double-strand break (DSB) repair via two pathways of HR that require end resection as an intermediate step, end resection of camptothecin (Cpt)-induced DNA damage, and RAD51 recruitment to ionizing radiation induced foci (IRIF), which requires end resection. Furthermore, XAB2 mediates specific aspects of the DNA damage response associated with end resection proficiency: CtIP hyperphosphorylation induced by Cpt and BRCA1 IRIF. XAB2 also promotes histone acetylation events linked to HR proficiency. From truncation mutation analysis, the capacity for XAB2 to promote HR correlates with its ability to form a complex with ISY1 and PRP19, which show a similar influence as XAB2 on HR. This XAB2 complex localizes to punctate structures consistent with interchromatin granules that show a striking adjacent-localization to the DSB marker γH2AX. In summary, we suggest that the XAB2 complex mediates DNA damage response events important for the end resection step of HR, and speculate that its adjacent-localization relative to DSBs marked by γH2AX is important for this function.

scholarly journals A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

2020 ◽  
Author(s):  
Chris R. Hackley
Keyword(s):  
Homologous Recombination ◽  
Long Range ◽  
Mammalian Cells ◽  
Strand Break ◽  
Dsb Repair ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Attractive Option ◽  
End Resection ◽  
Rate Limiting

AbstractCRISPR/Cas9 has revolutionized genetic engineering, however, the inability to control double strand break (DSB) repair has severely limited both therapeutic and academic applications. Many attempts have been made to control DSB repair choice, however particularly in the case of larger edits, none have been able to bypass the rate-limiting step of Homologous Recombination (HR): long-range 5’ end resection. Here we describe a novel set of Cas9 fusions, Cas9-HRs, designed to bypass the rate-limiting step of HR repair by simultaneously coupling initial and long-range end resection. Cas9-HRs can increase the rate HR by 2-2.5 fold and decrease cellular toxicity by 2-4 fold compared to Cas9 in mammalian cells with minimal apparent editing site bias, thus making Cas9-HRs an attractive option for applications demanding increased HDR rates for long inserts and/or reduced p53 pathway activation.SummaryA novel Cas9 fusion protein designed to increase HDR rates through bypassing the rate limiting step of homologous recombination repair.

scholarly journals Homologous recombination defects in Shwachman-Diamond syndrome and Diamond-Blackfan anemia

2020 ◽  
Author(s):  
Elif Asik ◽  
Nimrat Chatterjee ◽  
Alison A. Bertuch
Keyword(s):  
Homologous Recombination ◽  
Ribosome Biogenesis ◽  
Strand Break ◽  
Cancer Predisposition ◽  
Parp Inhibition ◽  
Dsb Repair ◽  
Diamond Blackfan Anemia ◽  
Dna Double Strand Break ◽  
Damage Response ◽  
Shwachman Diamond Syndrome

ABSTRACTShwachman-Diamond syndrome (SDS) and Diamond-Blackfan anemia (DBA) are ribosomopathies characterized by impaired hematopoiesis and cancer predisposition. The mechanisms underlying cancer predisposition in these disorders are not well understood. We found that LCLs derived from patients with SDS or DBA had a prolonged DNA damage response and hypersensitivity to ionizing radiation, suggesting impaired DNA double strand break (DSB) repair. Consistent with this, depletion of SBDS and RPS19, the most common etiologic factors in SDS and DBA, respectively, resulted in reduced homologous recombination (HR) in HCT116 and U2OS cells. Surprisingly, depletion of EFL1, which functions with SBDS in ribosome biogenesis, did not impair HR and depletion of eIF6, which restores ribosome joining in SBDS-depleted cells, did not rescue the HR defect associated with SBDS depletion. Instead, we found SBDS and RPS19 recruitment to sites of DSBs suggesting that SBDS and RPS19 have more proximate roles in regulating HR, independent of their ribosomal functions. We propose that reduced HR shifts DSB repair toward error-prone NHEJ and this may contribute to oncogenesis in SDS and DBA. Additionally, we found SBDS and RPS19 depleted cells were hypersensitive to PARP inhibition, potentially uncovering a therapeutic target for SDS- and DBA-associated malignancies.

scholarly journals RBX1 prompts degradation of EXO1 to limit the homologous recombination pathway of DNA double-strand break repair in G1 phase

2019 ◽  
Vol 27 (4) ◽  
pp. 1383-1397 ◽  
Author(s):  
Ying Xie ◽  
Yi-Ke Liu ◽  
Zong-Pei Guo ◽  
Hua Guan ◽  
Xiao-Dan Liu ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Expression Level ◽  
G1 Phase ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Elevated Expression ◽  
Recombination Pathway ◽  
Specific Degradation ◽  
End Resection

Abstract End resection of DNA double-strand breaks (DSBs) to form 3′ single-strand DNA (ssDNA) is critical to initiate the homologous recombination (HR) pathway of DSB repair. HR pathway is strictly limited in the G1-phase cells because of lack of homologous DNA as the templates. Exonuclease 1 (EXO1) is the key molecule responsible for 3′ ssDNA formation of DSB end resection. We revealed that EXO1 is inactivated in G1-phase cells via ubiquitination-mediated degradation, resulting from an elevated expression level of RING-box protein 1 (RBX1) in G1 phase. The increased RBX1 significantly prompted the neddylation of Cullin1 and contributed to the G1 phase-specific degradation of EXO1. Knockdown of RBX1 remarkedly attenuated the degradation of EXO1 and increased the end resection and HR activity in γ-irradiated G1-phase cells, as demonstrated by the increased formation of RPA32, BrdU, and RAD51 foci. And EXO1 depletion mitigated DNA repair defects due to RBX1 reduction. Moreover, increased autophosphorylation of DNA-PKcs at S2056 was found to be responsible for the higher expression level of the RBX1 in the G1 phase. Inactivation of DNA-PKcs decreased RBX1 expression, and simultaneously increased EXO1 expression and DSB end resection in G1-phase cells. This study demonstrates a new mechanism for restraining the HR pathway of DNA DSB repair in G1 phase via RBX1-prompted inactivation of EXO1.

Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes

2010 ◽  
Vol 431 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Eun-Jung Park ◽  
Shin-Kyoung Hur ◽  
Jongbum Kwon
Keyword(s):  
Chinese Hamster ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Direct Role ◽  
Dna Double Strand Break ◽  
Break Repair ◽  
Repair Defect

Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

scholarly journals GRB2 enforces homology-directed repair initiation by MRE11

2021 ◽  
Vol 7 (32) ◽  
pp. eabe9254
Author(s):  
Zu Ye ◽  
Shengfeng Xu ◽  
Yin Shi ◽  
Albino Bacolla ◽  
Aleem Syed ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Genome Stability ◽  
Synthetic Lethality ◽  
Mapk Pathway ◽  
Parp Inhibitor ◽  
Strand Break ◽  
Sh2 Domain ◽  
Dsb Repair ◽  
Homology Directed Repair ◽  
Genome Analyses

DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2’s role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.

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