Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection

Reginato, Giordano; Cannavó, Elda; Ćejka, Petr

doi:10.1101/gad.308254.117

Cited by 120 publications

(132 citation statements)

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“…Other studies also recently demonstrated yeast Mre11/Rad50/Xrs2(MRX) cleavage of various protein blocks including yeast Ku, which was also manganese-dependent in vitro (10,11). Here we observe approximately the same kinetics of DNA-PK release catalyzed by MRN and CtIP as we did previously, but in magnesium-only…”

Section: Main Textsupporting

confidence: 87%

“…At single-ended breaks during replication, the nuclease activity of Mre11 was shown to be important for removal of Ku (6), suggesting that MRN catalytic processing of ends during S phase is important for recombination-mediated repair of single-ended breaks. While we and others have shown that MRN endonuclease activity is specifically stimulated by protein blocks on DNA ends (8)(9)(10)(11), we considered the possibility that DNA-PK itself constitutes a powerful block to resection, and that DNA-PK may create a critical barrier to end processing at single-ended breaks where there is no other DNA end to participate in end joining.…”

Section: Main Textmentioning

confidence: 99%

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DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

Deshpande

Myler

Soniat

et al. 2018

Preprint

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Abstract:DNA-dependent Protein Kinase (DNA-PK) coordinates the repair of double-strand breaks through non-homologous end joining, the dominant repair pathway in mammalian cells. Breaks can also be resolved through homologous recombination during S/G 2 cell cycle phases, initiated by the Mre11-Rad50-Nbs1 (MRN) complex and CtIP-mediated resection of 5ʹ strands. The functions of DNA-PK are considered to be end-joining specific, but here we demonstrate that human DNA-PK also plays an important role in the processing of DNA double-strand breaks.Using ensemble biochemistry and single-molecule approaches, we show that the MRN complex in cooperation with CtIP is stimulated by DNA-PK to perform efficient endonucleolytic processing of DNA ends in physiological conditions. This activity requires both CDK and ATR phosphorylation of CtIP. These unexpected results could explain the absence of DNA-PK deletion mutations in the human population, as homologous recombination is an essential process in mammals.One sentence summary: An enzyme critical for non-homologous repair of DNA double-strand breaks also stimulates end processing for homologous recombination.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/395731 doi: bioRxiv preprint first posted online Aug. 19, 2018; 3 Main Text:DNA-dependent Protein Kinase consists of a catalytic kinase subunit (DNA-PKcs) and the DNA end-binding heterodimer of Ku70 and Ku80 (Ku). Together, these proteins form an end recognition complex (DNA-PK) that binds to DNA double-strand breaks within seconds of break formation (1). DNA-PK promotes the ligation of two DNA ends by ligase IV, aided by the accessory factors XLF, XRCC4, and APLF, and in some cases aided by limited end processing (1,2). The recognition and repair of DNA breaks by the end-joining machinery is generally considered to be the first and rapid phase of DNA repair, occurring within ~30 minutes of DNA damage, while homologous recombination is specific to the S and G 2 phases of the cell cycle and occurs over a longer time frame (3,4). DNA-PK is present at micromolar concentrations in cells (5) and binds DNA ends in all cell cycle phases, even during S phase at single-ended breaks (6). The MRN complex regulates the initiation of DNA end processing prior to homologous recombination by catalyzing the initial 5ʹ strand processing at blocked DNA ends and by recruiting long-range nucleases Exo1 and Dna2 (4, 7). At single-ended breaks during replication, the nuclease activity of Mre11 was shown to be important for removal of Ku (6), suggesting that MRN catalytic processing of ends during S phase is important for recombination-mediated repair of single-ended breaks. While we and others have shown that MRN endonuclease activity is specifically stimulated by protein blocks on DNA ends (8-11), we considered the possibility that DNA-P...

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Section: Main Textsupporting

confidence: 87%

Section: Main Textmentioning

confidence: 99%

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

Deshpande

Myler

Soniat

et al. 2018

Preprint

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“…This is achieved by a conserved interaction between the MRN subunit NBS1 (Xrs2 in Saccharomyces cerevisiae) and ATM (Tel1 in yeast). MRE11 is likely the first nuclease to resect DSBs, which is particularly important for the processing of breaks with secondary DNA structures or protein adducts such as stalled topoisomerases or Ku (Shibata et al, 2014;Anand et al, 2016;Deshpande et al, 2016;Wang et al, 2017;Reginato et al, 2018). Beyond ATM, NBS1 also interacts with MDC1, which binds phosphorylated proteins, including H2AX (cH2AX), to amplify the DNA damage signaling beyond the vicinity of the DSB (Goldberg et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

“…In bacteria, the MRE11-RAD50-like SbcC-SbcD complex endonucleolytically cleaves DNA near blocked DNA ends (Connelly et al, 2003). In S. cerevisiae, Sae2 phosphorylated by CDK and other kinases has a critical function to promote the Mre11-Rad50 endonuclease near protein blocks (Huertas et al, 2008;Cannavo & Cejka, 2014;Wang et al, 2017;Reginato et al, 2018), while Xrs2 per se is not essential for the DNA clipping reaction in vitro (Oh et al, 2016), but may have a stimulatory function (Wang et al, 2017). In S. cerevisiae, Sae2 phosphorylated by CDK and other kinases has a critical function to promote the Mre11-Rad50 endonuclease near protein blocks (Huertas et al, 2008;Cannavo & Cejka, 2014;Wang et al, 2017;Reginato et al, 2018), while Xrs2 per se is not essential for the DNA clipping reaction in vitro (Oh et al, 2016), but may have a stimulatory function (Wang et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation

et al. 2019

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DNA end resection initiates DNA double‐strand break repair by homologous recombination. MRE11‐RAD50‐NBS1 and phosphorylated CtIP perform the first resection step via MRE11‐catalyzed endonucleolytic DNA cleavage. Human NBS1, more than its homologue Xrs2 in Saccharomyces cerevisiae, is crucial for this process, highlighting complex mechanisms that regulate the MRE11 nuclease in higher eukaryotes. Using a reconstituted system, we show here that NBS1, through its FHA and BRCT domains, functions as a sensor of CtIP phosphorylation. NBS1 then activates the MRE11‐RAD50 nuclease through direct physical interactions with MRE11. In the absence of NBS1, MRE11‐RAD50 exhibits a weaker nuclease activity, which requires CtIP but not strictly its phosphorylation. This identifies at least two mechanisms by which CtIP augments MRE11: a phosphorylation‐dependent mode through NBS1 and a phosphorylation‐independent mode without NBS1. In support, we show that limited DNA end resection occurs in vivo in the absence of the FHA and BRCT domains of NBS1. Collectively, our data suggest that NBS1 restricts the MRE11‐RAD50 nuclease to S‐G2 phase when CtIP is extensively phosphorylated. This defines mechanisms that regulate the MRE11 nuclease in DNA metabolism.

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“…In both yeast and mammals, the current model for resection posits that Sae2 stimulates the endonuclease activity of Mre11 to generate an endonucleolytic cleavage of the 5 0 -terminated strands at both DSB ends (Cannavo & Cejka, 2014). The resulting nicks are entry sites for Exo1 and Dna2 nucleases that degrade long tracts of DNA in the 5 0 -3 0 direction (Mimitou & Symington, 2008;Zhu et al, 2008;Cejka et al, 2010;Niu et al, 2010;Nimonkar et al, 2011), coupled with 3 0 -5 0 exonuclease of Mre11 back toward the DNA end (Garcia et al, 2011;Shibata et al, 2014;Reginato et al, 2017;Wang et al, 2017). While Exo1 degrades the 5 0 -terminated strands within duplex DNA (Tran et al, 2002), Dna2 cleaves ssDNA flaps adjoining a duplex DNA.…”

Section: Introductionmentioning

confidence: 99%

The MRX complex regulates Exo1 resection activity by altering DNA end structure

et al. 2018

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Homologous recombination is triggered by nucleolytic degradation (resection) of DNA double-strand breaks (DSBs). DSB resection requires the Mre11-Rad50-Xrs2 (MRX) complex, which promotes the activity of Exo1 nuclease through a poorly understood mechanism. Here, we describe the Mre11-R10T mutant variant that accelerates DSB resection compared to wild-type Mre11 by potentiating Exo1-mediated processing. This increased Exo1 resection activity leads to a decreased association of the Ku complex to DSBs and an enhanced DSB resection in G1, indicating that Exo1 has a direct function in preventing Ku association with DSBs. Molecular dynamics simulations show that rotation of the Mre11 capping domains is able to induce unwinding of double-strand DNA (dsDNA). The R10T substitution causes altered orientation of the Mre11 capping domain that leads to persistent melting of the dsDNA end. We propose that MRX creates a specific DNA end structure that promotes Exo1 resection activity by facilitating the persistence of this nuclease on the DSB ends, uncovering a novel MRX function in DSB resection.

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Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection

Cited by 120 publications

References 40 publications

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

NBS1 promotes the endonuclease activity of the MRE11‐RAD50 complex by sensing CtIP phosphorylation

The MRX complex regulates Exo1 resection activity by altering DNA end structure

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