The MRN complex and topoisomerase IIIa–RMI1/2 synchronize DNA resection motor proteins
2022; Elsevier BV; Volume: 299; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2022.102802
ISSN1083-351X
AutoresMichael M. Soniat, Giaochau Nguyen, Hung‐Che Kuo, Ilya J. Finkelstein,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoDNA resection—the nucleolytic processing of broken DNA ends—is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11–RAD50–NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)–RMI1/2 during long-range DNA resection. BLM partners with TOP3A–RMI1/2 to form the BTRR (BLM–TOP3A–RMI1/2) complex (or BLM dissolvasome). We determined that TOP3A–RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A–RMI1/2 regulates the helicase activity of BLM to promote efficient DNA repair. DNA resection—the nucleolytic processing of broken DNA ends—is the first step of homologous recombination. Resection is catalyzed by the resectosome, a multienzyme complex that includes bloom syndrome helicase (BLM), DNA2 or exonuclease 1 nucleases, and additional DNA-binding proteins. Although the molecular players have been known for over a decade, how the individual proteins work together to regulate DNA resection remains unknown. Using single-molecule imaging, we characterized the roles of the MRE11–RAD50–NBS1 complex (MRN) and topoisomerase IIIa (TOP3A)–RMI1/2 during long-range DNA resection. BLM partners with TOP3A–RMI1/2 to form the BTRR (BLM–TOP3A–RMI1/2) complex (or BLM dissolvasome). We determined that TOP3A–RMI1/2 aids BLM in initiating DNA unwinding, and along with MRN, stimulates DNA2-mediated resection. Furthermore, we found that MRN promotes the association between BTRR and DNA and synchronizes BLM and DNA2 translocation to prevent BLM from pausing during resection. Together, this work provides direct observation of how MRN and DNA2 harness the BTRR complex to resect DNA efficiently and how TOP3A–RMI1/2 regulates the helicase activity of BLM to promote efficient DNA repair. Homologous recombination (HR) is one of two major eukaryotic dsDNA break (DSB) repair pathways. HR uses the intact sister chromatid during the S/G2 phase to promote error-free repair of DSBs (1Jasin M. Rothstein R. Repair of strand breaks by homologous recombination.Cold Spring Harb. Perspect. Biol. 2013; 5: a012740Crossref PubMed Scopus (594) Google Scholar, 2Mathiasen D.P. Lisby M. 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In later stages of HR, following RAD51 loading and strand invasion, BTRR is critical for the branch migration of Holliday junctions to form a hemicatenane, followed by decatenation by TOP3A–RMI1/2, resulting in noncrossovers (46Bizard A.H. Hickson I.D. The dissolution of double Holliday junctions.Cold Spring Harb. Perspect. Biol. 2014; 6: a016477Crossref PubMed Scopus (136) Google Scholar, 47Wu L. Hickson I.D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination.Nature. 2003; 426: 870-874Crossref PubMed Scopus (883) Google Scholar). Though the role of TOP3A–RMI1/2 is well defined in the later stages of HR, their precise roles in DNA resection remain unclear. For example, a catalytically inactive TOP3A mutant stimulates long-range DNA resection in vitro, suggesting that TOP3A–RMI1/2 may play a nonenzymatic role in resectosome assembly and/or translocation (20Niu H. Chung W.-H. Zhu Z. Kwon Y. Zhao W. Chi P. et al.Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.Nature. 2010; 467: 108-111Crossref PubMed Scopus (294) Google Scholar, 44Daley J.M. Chiba T. Xue X. Niu H. Sung P. Multifaceted role of the Topo IIIα-RMI1-RMI2 complex and DNA2 in the BLM-dependent pathway of DNA break end resection.Nucl. Acids Res. 2014; 42: 11083-11091Crossref PubMed Scopus (54) Google Scholar). Here, we use single-molecule fluorescence imaging to decipher the functions of individual resectosome components during DNA resection. Both MRN and TOP3A–RMI1/2 help BLM to initiate DNA unwinding. MRN and TOP3A–RMI1/2 also stimulate DNA2-mediated resection. Finally, MRN synchronizes the translocation speeds of BLM and DNA2 to prevent BLM pausing. We reveal that MRN and TOP3A–RMI1/2 are regulatory resectosome components that initiate DNA resection and synchronize the individual motors during kilobase-long DNA processing. To understand how MRN and TOP3A–RMI1/2 regulate DNA processing, we adapted our single-molecule resection assay to quantify the movement of DNA2 or EXO1 in complex with MRN and BTRR (Fig. 1) (28Myler L.R. Gallardo I.F. Soniat M.M. Deshpande R.A. Gonzalez X.B. Kim Y. et al.Single-molecule imaging reveals how mre11-rad50-Nbs1 initiates DNA break repair.Mol. Cell. 2017; 67: 891-898.e4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 48Myler L.R. Gallardo I.F. Zhou Y. Gong F. Yang S.-H. Wold M.S. et al.Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: e1170-e1179Crossref PubMed Scopus (65) Google Scholar, 49Soniat M.M. Myler L.R. Schaub J.M. Kim Y. Gallardo I.F. Finkelstein I.J. Next-generation DNA curtains for single-molecule studies of homologous recombination.Meth. Enzymol. 2017; 592: 259-281Crossref PubMed Scopus (20) Google Scholar, 50Soniat M.M. Myler L.R. Kuo H.-C. Paull T.T. Finkelstein I.J. RPA phosphorylation inhibits DNA resection.Mol. Cell. 2019; 75: 145-153.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). We purified RMI1/2 with an N-terminal FLAG epitope on the RMI2 subunit. TOP3A–RMI1/2 was reconstituted by mixing TOP3A and RMI1/2 in a 1:3 ratio, followed by size-exclusion chromatography. These three proteins formed a stable complex that eluted as a single peak on a Superose 6 column (GE Healthcare) (Fig. S1A). TOP3A–RMI1/2 was then mixed with BLM to assemble the BTRR resectosome and labeled with fluorescent anti-FLAG antibodies (targeting RMI2 as described previously). Biotinylated MRN was conjugated with streptavidin quantum dots (QDs) that emit in spectrally distinct channels (Fig. S1, A and B). We have previously shown that fluorescently labeled MRN retains its biochemical activities, including diffusion along nucleosomal DNA and nucleolytic removal of Ku from DNA ends (28Myler L.R. Gallardo I.F. Soniat M.M. Deshpande R.A. Gonzalez X.B. Kim Y. et al.Single-molecule imaging reveals how mre11-rad50-Nbs1 initiates DNA break repair.Mol. Cell. 2017; 67: 891-898.e4Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 51Deshpande R.A. Myler L.R. Soniat M.M. Makharashvili N. Lee L. Lees-Miller S.P. et al.DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP.Sci. Adv. 2020; 6eaay0922Crossref Scopus (64) Google Scholar, 52Myler L.R. Soniat M.M. Zhang X. Deshpande R.A. Paull T.T. Finkelstein I.J. purification and biophysical characterization of the mre11-rad50-Nbs1 complex.Met. Mol. Biol. 2019; 2004: 269-287Crossref PubMed Scopus (2) Google Scholar). In addition, QD-labeled MRN does not prevent interactions with other resectosome components, such as EXO1, consistent with in vivo and ensemble in vitro assays with unlabeled MRN. For the single-molecule resection assay, 48.5-kb-long dsDNAs with biotin on one end and a 78 nt 3′-overhang on the opposite end are organized on the surface of a microfluidic flow cell (Fig. 1B) (49Soniat M.M. Myler L.R. Schaub J.M. Kim Y. Gallardo I.F. Finkelstein I.J. Next-generation DNA curtains for single-molecule studies of homologous recombination.Meth. Enzymol. 2017; 592: 259-281Crossref PubMed Scopus (20) Google Scholar, 53Gallardo I.F. Pasupathy P. Brown M. Manhart C.M. Neikirk D.P. Alani E. et al.High-throughput universal DNA curtain arrays for single-molecule fluorescence imaging.Langmuir. 2015; 31: 10310-10317Crossref PubMed Scopus (41) Google Scholar). Fluorescent BTRR complex is incubated with MRN and DNA2 or EXO1 before being injected into flow cells for single-molecule imaging (Fig. S1C). As expected, MRN and BTRR in complex with DNA2 and EXO1 bound the free DNA ends and resected the DNA in the presence of 1 nM RPA (Fig. 1C). The MRN–BTRR–DNA2 complex resected DNA for 18 ± 6 kb (mean ± SD; n = 82) with a velocity of 18 ± 11 bp s−1. Omitting either TOP3A–RMI1/2 or MRN decreased BLM–DNA2 velocity for approximately two-fold (BTRR–DNA2: 9 ± 6 bp s−1, n = 94 molecules; MRN–BLM–DNA2: 9 ± 6 bp s−1, n = 30) and decreased processivity by 1.4-fold (BTRR–DNA2: 13 ± 5 kb; MRN–BLM–DNA2: 12 ± 5 kb) (Fig. 1, C–E and Table S1). Our results with the human resectosome are consistent with the stimulation of S. cerevisiae Sgs1–Dna2 by MRX and Top3–Rmi1 (19Cejka P. Cannavo E. Polaczek P. Masuda-Sasa T. Pokharel S. Campbell J.L. et al.DNA end resection by Dna2-Sgs1-RPA and its stimulation by Top3-Rmi1 and Mre11-Rad50-Xrs2.Nature. 2010; 467: 112-116Crossref PubMed Scopus (350) Google Scholar, 20Niu H. Chung W.-H. Zhu Z. Kwon Y. Zhao W. Chi P. et al.Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.Nature. 2010; 467: 108-111Crossref PubMed Scopus (294) Google Scholar). In contrast, the addition of MRN and TOP3A–RMI1/2 to BLM–EXO1 resectosomes did not change the processivity or velocity (∼12 ± 7 kb; ∼13 ± 10 bp s−1; n > 50 for all conditions), suggesting that MRN and TOP3A–RMI1/2 selectively regulate DNA2-mediated resection (Fig. 1, F and G and Table S1). In addition to its nucleolytic activity, DNA2 also encodes a 5’→3′ helicase domain that can unwind kilobases of dsDNA (54Levikova M. Klaue D. Seidel R. Cejka P. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1992-2001Crossref PubMed Scopus (45) Google Scholar, 55Levikova M. Pinto C. Cejka P. The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection.Genes Dev. 2017; 31: 493-502Crossref PubMed Scopus (36) Google Scholar, 56Pinto C. Kasaciunaite K. Seidel R. Cejka P. 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Furthermore, a helicase-deficient (hd) DNA2(K654R) mutant decreased resection processivity and velocity (processivity: 13 ± 6 kb; velocity: 11 ± 7 bp s−1, n = 76). Omitting TOP3A–RMI1/2 and MRN abrogated the negative effects of DNA2(K654R) on resection processivity and velocity (14 ± 8 kb; 12 ± 8 bp s−1, n = 42) (Fig. S2, A and B). We repeated the resection experiments with an hd BLM(K695A) mutant on both 5′-overhang and 3′-overhang DNA substrates but did not see long-distance motor activity beyond our ∼500 bp resolution (Fig. S2C). This indicates that the helicase activity of BLM provides a 5′-flap for DNA2. In addition, BLM prevents the initiation of long-range helicase activity by DNA2, even with a 12-nt 5′-overhang. These results are consistent with our previous study that showed that hd BLM also blocks nuclease-dead DNA2 from unwinding DNA (50Soniat M.M. Myler L.R. Kuo H.-C. Paull T.T. Finkelstein I.J. RPA phosphorylation inhibits DNA resection.Mol. Cell. 2019; 75: 145-153.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These results show that both the nuclease and helicase activity of DNA2 are required for rapid long-range DNA processing. Since TOP3A–RMI1/2 and MRN stimulate the BLM–DNA2 resectosome, we tested whether either TOP3A–RMI1/2 and/or MRN can stimulate DNA2 alone. MRN recruits DNA2 to DSBs in human cells but does not affect DNA2 nuclease activity in vitro (30Nimonkar A.V. Genschel J. Kinoshita E. Polaczek P. Campbell J.L. Wyman C. et al.BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.Genes Dev. 2011; 25: 350-362Crossref PubMed Scopus (519) Google Scholar). Consistent with this report, 87% of DNA2 molecules colocalized with MRN (n = 97/112) at free DNA ends (Fig. 2, C and D). We saw a more modest 65% of TOP3A–RMI1/2 complexes (n = 22/34) colocalizing with DNA2. DNA2 did not translocate beyond our spatial resolution of ∼500 bp with MRN and TOP3A–RMI1/2 (together or independently) in the presence of RPA. Previous studies showed that suppression of the nuclease activity of DNA2 stimulates processive helicase activity on DNA substrates containing a 5′-flap in the presence of RPA (54Levikova M. Klaue D. Seidel R. Cejka P. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1992-2001Crossref PubMed Scopus (45) Google Scholar, 55Levikova M. Pinto C. Cejka P. The motor activity of DNA2 functions as an ssDNA translocase to promote DNA end resection.Genes Dev. 2017; 31: 493-502Crossref PubMed Scopus (36) Google Scholar, 56Pinto C. Kasaciunaite K. Seidel R. Cejka P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.Elife. 2016; https://doi.org/10.7554/eLife.18574Crossref Scopus (51) Google Scholar). To observe the helicase activity, we monitored nuclease-deficient DNA2(D277A) on DNA substrates containing 12 nt 5′-overhang in the presence of 1 nM RPA, similar to previous studies (54Levikova M. Klaue D. Seidel R. Cejka P. Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E1992-2001Crossref PubMed Scopus (45) Google Scholar, 56Pinto C. Kasaciunaite K. Seidel R. Cejka P. Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases.Elife. 2016; https://doi.org/10.7554/eLife.18574Crossref Scopus (51) Google Scholar, 59Ceppi I. Howard S.M. Kasaciunaite K. Pinto C. Anand R. Seidel R. et al.CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection.Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 8859-8869Crossref PubMed Scopus (37) Google Scholar). DNA2 was a processive helicase, motoring ∼4 ± 2 kb with a velocity of 5 ± 3 bp s−1 (n = 23 molecules) (Fig. 2, E–G and Table S2). The addition of MRN, TOP3A–RMI1/2, or both together had no effect on the processivity of DNA2. However, adding either MRN or TOP3A–RMI1/2 decreased the velocity of DNA2 by ∼1.4-fold relative to DNA2(D277A) alone. In contrast, the addition of both MRN and TOP3A–RMI1/2 restored helicase activity to that of DNA2 alone. These results are broadly consistent with a model where MRN and TOP3A–RMI1/2 help DNA2 engage free DNA ends but do not stimulate its nuclease or motor activities. Next, we investigated how TOP3A–RMI1/2 regulates BLM helicase. BLM was fluorescently labeled via a fluorescent anti-hemagglutinin (HA) antibody directed to an N-terminal HA epitope, as we described previously (50Soniat M.M. Myler L.R. Kuo H.-C. Paull T.T. Finkelstein I.J. RPA phosphorylation inhibits DNA resection.Mol. Cell. 2019; 75: 145-153.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). TOP3A–RMI1/2 colocalized with BLM at the free DNA ends (Fig. 3A). We recently showed that RPA aids BLM in initiating helicase activity from 3′-ssDNA overhangs (50Soniat M.M. Myler L.R. Kuo H.-C. Paull T.T. Finkelstein I.J. RPA phosphorylation inhibits DNA resection.Mol. Cell. 2019; 75: 145-153.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). When RPA is omitted, only ∼30% of BLM molecules initiate translocation. However, adding TOP3A–RMI1/2 increases the number of translocating BLM molecules ∼2.3-fold (77%; n = 86/111) (Fig. 3B). TOP3A was sufficient to recapitulate most of this stimulation (62%; n = 83/133), whereas adding RMI1/2 alone slightly decreased the number of translocating BLM molecules (19%; n = 34/182). Finally, adding RPA did not further stimulate the number of translocating BTRR complexes (83%; n = 67/81). Surprisingly, although TOP3A–RMI1/2 initiated more BLM helicases, it also decreased the velocity of BLM approximately two-fold (14 ± 11 bp s−1; n = 86) and slightly reduced processivity ∼1.2-fold (14 ± 8 kb) (Fig.
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