The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

Liao, Shuren; Tammaro, Margaret; Yan, Hong

doi:10.1093/nar/gkw274

Cited by 31 publications

(35 citation statements)

References 72 publications

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“…Purified proteins were aliquoted to 3 μl, frozen in liquid nitrogen and stored at –80°C. Expression and purification of wild-type (WT) MRN complex and a nuclease dead-mutant complex containing Mre11(H130N) were described previously (80,88). Expression and purification of WT RPA complex and the RPA (1NΔ) mutant complex were described previously (61).…”

Section: Methodsmentioning

confidence: 99%

“…Consistent with this idea, it has been shown that Sae2 and MRX are not essential for resection and downstream HR at clean DSBs in yeast (76–79). In the Xenopus cytosolic extract the nuclease activity of MRN is dispensable for the overall resection of clean DSBs, which is in sharp contrast to 5′ blocked ends where the nuclease activity of MRN is absolutely essential (80). Likewise, the catalytic function of CtIP has also been shown to be dispensable for resection of clean DSBs in human cells (43).…”

Section: Introductionmentioning

confidence: 99%

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Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5′ strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5′ strand DNA ∼10–20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5′ strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP–MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Dna2 initiates resection at clean DNA double-strand breaks

Paudyal

Yan

et al. 2017

Nucleic Acids Research

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“…Key factors regulating pathway choice for DSB repair are the cell cycle (116) and the nature of DNA ends (69). Whereas NHEJ-based repair pathways are active throughout the cell cycle, HR requires a DNA template for the repair and is active in only the S and G2 phases, during which the homologous sister chromosome is present.…”

Section: Mrn and Double-strand Break Repairmentioning

confidence: 99%

“…Whereas NHEJ-based repair pathways are active throughout the cell cycle, HR requires a DNA template for the repair and is active in only the S and G2 phases, during which the homologous sister chromosome is present. The nature of the DSB ends also regulates the repair pathway choice (69). If the DSB ends are free of protein adducts and damaged nucleotides, they are likely channeled through NHEJ for repair in human cells.…”

Section: Mrn and Double-strand Break Repairmentioning

confidence: 99%

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

Syed

Tainer

2018

Annu. Rev. Biochem.

324

345

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Genomic instability in disease and its fidelity in health depend on the DNA damage response (DDR), regulated in part from the complex of meiotic recombination 11 homolog 1 (MRE11), ATP-binding cassette–ATPase (RAD50), and phosphopeptide-binding Nijmegen breakage syndrome protein 1 (NBS1). The MRE11–RAD50–NBS1 (MRN) complex forms a multifunctional DDR machine. Within its network assemblies, MRN is the core conductor for the initial and sustained responses to DNA double-strand breaks, stalled replication forks, dysfunctional telomeres, and viral DNA infection. MRN can interfere with cancer therapy and is an attractive target for precision medicine. Its conformations change the paradigm whereby kinases initiate damage sensing. Delineated results reveal kinase activation, posttranslational targeting, functional scaffolding, conformations storing binding energy and en-abling access, interactions with hub proteins such as replication protein A (RPA), and distinct networks at DNA breaks and forks. MRN biochemistry provides prototypic insights into how it initiates, implements, and regulates multifunctional responses to genomic stress.

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“…The channeling of DSB repair intermediates to HR requires CtIP. That CtIP stimulates Mre11 endonuclease activity at DNA-protein crosslinks [17,94] suggests a mechanism for releasing Ku from DNA ends, thereby promoting resection and HR repair over DNA end protection and NHEJ repair [71,95]. …”

Section: Ctip Is a Regulatory Hub For Dsb Repair Pathway Choicementioning

confidence: 99%

CtIP/Ctp1/Sae2, molecular form fit for function

Andres¹,

Williams²

2017

DNA Repair

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Vertebrate CtIP, and its fission yeast (Ctp1), budding yeast (Sae2) and plant (Com1) orthologs have emerged as key regulatory molecules in cellular responses to DNA double strand breaks (DSBs). By modulating the nucleolytic 5′-3′ resection activity of the Mre11/Rad50/Nbs1 (MRN) DSB repair processing and signaling complex, CtIP/Ctp1/Sae2/Com1 is integral to the channeling of DNA double strand breaks through DSB repair by homologous recombination (HR). Nearly two decades since its discovery, emerging new data are defining the molecular underpinnings for CtIP DSB repair regulatory activities. CtIP homologs are largely intrinsically unstructured proteins comprised of expanded regions of low complexity sequence, rather than defined folded domains typical of DNA damage metabolizing enzymes and nucleases. A compact structurally conserved N-terminus forms a functionally critical tetrameric helical dimer of dimers (THDD) region that bridges CtIP oligomers, and is flexibly appended to a conserved C-terminal Sae2-homology DNA binding and DSB repair pathway choice regulatory hub which influences nucleolytic activities of the MRN core nuclease complex. The emerging evidence from structural, biophysical, and biological studies converges on CtIP having functional roles in DSB repair that include: 1) dynamic DNA strand coordination through direct DNA binding and DNA bridging activities, 2) MRN nuclease complex cofactor functions that direct MRN endonucleolytic cleavage of protein-blocked DSB ends and 3) acting as a protein binding hub targeted by the cell cycle regulatory apparatus, which influences CtIP expression and activity via layers of post-translational modifications, protein-protein interactions and DNA binding.

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The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

Cited by 31 publications

References 72 publications

Dna2 initiates resection at clean DNA double-strand breaks

Dna2 initiates resection at clean DNA double-strand breaks

The MRE11–RAD50–NBS1 Complex Conducts the Orchestration of Damage Signaling and Outcomes to Stress in DNA Replication and Repair

CtIP/Ctp1/Sae2, molecular form fit for function

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