Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3′ single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1–dependent or –independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1–Dna2–Top3-Rmi1–RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

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A role for RNA and DNA:RNA hybrids in the modulation of DNA repair by homologous recombination

10.1101/255976 ◽

2018 ◽

Author(s):

Giuseppina D’Alessandro ◽

Marek Adamowicz ◽

Donna Whelan ◽

Sean Michael Howard ◽

Corey Winston Jones-Weinert ◽

...

Keyword(s):

Homologous Recombination ◽

Dna Lesions ◽

Cycle Phase ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna End Resection ◽

Brca1 Brca2 ◽

Rnase H2 ◽

End Resection ◽

Dna Ends

AbstractDNA double-strand breaks (DSBs) are toxic DNA lesions which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1 and promote its recruitment to DSBs. We also show that RNase H2 is in a complex with the HR proteins BRCA1, PALB2, BRCA2, and RAD51, and that it localizes to DSBs in the S/G2 cell-cycle phase. BRCA2 controls DNA:RNA hybrid levels at DSBs by mediating RNase H2 recruitment and, therefore, hybrids degradation. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.

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Human SIRT6 Promotes DNA End Resection Through CtIP Deacetylation

Science ◽

10.1126/science.1192049 ◽

2010 ◽

Vol 329 (5997) ◽

pp. 1348-1353 ◽

Cited By ~ 261

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.

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CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkz639 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9160-9179 ◽

Cited By ~ 6

Author(s):

Soon Young Hwang ◽

Mi Ae Kang ◽

Chul Joon Baik ◽

Yejin Lee ◽

Ngo Thanh Hang ◽

...

Keyword(s):

Homologous Recombination ◽

Critical Role ◽

Control Point ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

C Terminus ◽

Dna End Resection ◽

End Resection

Abstract The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

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The internal region of CtIP negatively regulates DNA end resection

Nucleic Acids Research ◽

10.1093/nar/gkaa273 ◽

2020 ◽

Vol 48 (10) ◽

pp. 5485-5498 ◽

Cited By ~ 2

Author(s):

Sean Michael Howard ◽

Ilaria Ceppi ◽

Roopesh Anand ◽

Roger Geiger ◽

Petr Cejka

Keyword(s):

Homologous Recombination ◽

Regulatory Function ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Internal Region ◽

Dna End Resection ◽

End Resection

Abstract DNA double-strand breaks are repaired by end-joining or homologous recombination. A key-committing step of recombination is DNA end resection. In resection, phosphorylated CtIP first promotes the endonuclease of MRE11–RAD50–NBS1 (MRN). Subsequently, CtIP also stimulates the WRN/BLM–DNA2 pathway, coordinating thus both short and long-range resection. The structure of CtIP differs from its orthologues in yeast, as it contains a large internal unstructured region. Here, we conducted a domain analysis of CtIP to define the function of the internal region in DNA end resection. We found that residues 350–600 were entirely dispensable for resection in vitro. A mutant lacking these residues was unexpectedly more efficient than full-length CtIP in DNA end resection and homologous recombination in vivo, and consequently conferred resistance to lesions induced by the topoisomerase poison camptothecin, which require high MRN–CtIP-dependent resection activity for repair. This suggested that the internal CtIP region, further mapped to residues 550–600, may mediate a negative regulatory function to prevent over resection in vivo. The CtIP internal deletion mutant exhibited sensitivity to other DNA-damaging drugs, showing that upregulated resection may be instead toxic under different conditions. These experiments together identify a region within the central CtIP domain that negatively regulates DNA end resection.

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Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA Repair ◽

10.1016/j.dnarep.2011.12.007 ◽

2012 ◽

Vol 11 (3) ◽

pp. 310-316 ◽

Cited By ~ 52

Author(s):

Zhengping Shao ◽

Anthony J. Davis ◽

Kazi R. Fattah ◽

Sairei So ◽

Jingxin Sun ◽

...

Keyword(s):

Homologous Recombination ◽

Dna End Resection ◽

End Resection ◽

Dna Ends

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Super-resolution mapping of cellular double-strand break resection complexes during homologous recombination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021963118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2021963118

Author(s):

Donna R. Whelan ◽

Eli Rothenberg

Keyword(s):

Homologous Recombination ◽

Single Molecule ◽

Protein A ◽

Super Resolution ◽

Initial Step ◽

Double Strand Breaks ◽

Homology Search ◽

Major Pathway ◽

Strand Breaks

Homologous recombination (HR) is a major pathway for repair of DNA double-strand breaks (DSBs). The initial step that drives the HR process is resection of DNA at the DSB, during which a multitude of nucleases, mediators, and signaling proteins accumulates at the damage foci in a manner that remains elusive. Using single-molecule localization super-resolution (SR) imaging assays, we specifically visualize the spatiotemporal behavior of key mediator and nuclease proteins as they resect DNA at single-ended double-strand breaks (seDSBs) formed at collapsed replication forks. By characterizing these associations, we reveal the in vivo dynamics of resection complexes involved in generating the long single-stranded DNA (ssDNA) overhang prior to homology search. We show that 53BP1, a protein known to antagonize HR, is recruited to seDSB foci during early resection but is spatially separated from repair activities. Contemporaneously, CtBP-interacting protein (CtIP) and MRN (MRE11-RAD51-NBS1) associate with seDSBs, interacting with each other and BRCA1. The HR nucleases EXO1 and DNA2 are also recruited and colocalize with each other and with the repair helicase Bloom syndrome protein (BLM), demonstrating multiple simultaneous resection events. Quantification of replication protein A (RPA) accumulation and ssDNA generation shows that resection is completed 2 to 4 h after break induction. However, both BRCA1 and BLM persist later into HR, demonstrating potential roles in homology search and repair resolution. Furthermore, we show that initial recruitment of BRCA1 and removal of Ku are largely independent of MRE11 exonuclease activity but dependent on MRE11 endonuclease activity. Combined, our observations provide a detailed description of resection during HR repair.

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Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

Biochemical Society Transactions ◽

10.1042/bst20180518 ◽

2019 ◽

Vol 47 (6) ◽

pp. 1609-1619 ◽

Cited By ~ 5

Author(s):

Qian Wu

Keyword(s):

Single Molecule ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Structural Mechanism ◽

Strand Breaks ◽

Single Molecule Studies ◽

Non Homologous End Joining ◽

Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

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XLF acts as a flexible connector during non-homologous end joining

10.1101/2020.07.29.227033 ◽

2020 ◽

Author(s):

Sean M. Carney ◽

Andrew T. Moreno ◽

Sadie C. Piatt ◽

Metztli Cisneros-Aguirre ◽

Felicia Wednesday Lopezcolorado ◽

...

Keyword(s):

Single Molecule ◽

Critical Factor ◽

Tail Length ◽

Binding Motif ◽

Dna Double Strand Breaks ◽

End Joining ◽

Synaptic Complex ◽

Intrinsically Disordered ◽

Non Homologous End Joining ◽

Dna Ends

AbstractNon-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

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