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• W2037004712 abstract "DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast. DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast. DNA double-strand breaks (DSB) 3The abbreviations used are: DSBDNA double strand breakNHEJnon-homologous end-joiningHRhomologous recombinationDNA-PKcsDNA-dependent protein kinase catalytic subunitExo1exonuclease 1MRMre11/Rad50MRNMre11/Rad50/Nbs1MRXMre11/Rad50/Xrs2yKuyeast KuF-DNAforked DNAntnucleotide(s). can be generated spontaneously by endogenous sources, such as DNA replication-associated errors and by products of cellular metabolism, or exogenous agents including ionizing irradiation and radiomimetic chemicals (1Lindahl T. Prigent C. Barnes D.E. Lehmann A.R. Satoh M.S. Roberts E. Nash R.A. Robins P. Daly G. DNA joining in mammalian cells.Cold Spring Harb. Symp. Quant. Biol. 1993; 58: 619-624Crossref PubMed Scopus (16) Google Scholar). Upon exposure to DSBs, prompt and precise DSB repair is critical because unrepaired DSBs can result in genomic instability, cell death, and tumorigenesis (2Burma S. Chen B.P. Chen D.J. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair. 2006; 5: 1042-1048Crossref PubMed Scopus (310) Google Scholar). There are two major DSB repair pathways, homologous recombination (HR) and nonhomologous end joining (NHEJ) (3Pastink A. Eeken J.C. Lohman P.H. Genomic integrity and the repair of double strand DNA breaks.Mutat. Res. 2001; 480: 37-50Crossref PubMed Scopus (202) Google Scholar). HR involves a series of steps including damage sensing, DNA resection, strand invasion, DNA synthesis, and ligation. HR is likely initiated when the Mre11/Rad50/Nbs1 (MRN) complex recognizes the DSB and recruits CtIP and Exo1 to mediate DNA resection. Resection of the DSB ends generates 3′-single strand DNA overhangs that are bound with RPA and then replaced by Rad51 for homologous template invasion and HR completion (4Sun H. Treco D. Szostak J.W. Extensive 3′-overhanging, single-stranded DNA associated with the meiosis-specific double strand breaks at the ARG4 recombination initiation site.Cell. 1991; 64: 1155-1161Abstract Full Text PDF PubMed Scopus (424) Google Scholar, 5White M.B. Word C.J. Humphries C.G. Blattner F.R. Tucker P.W. Immunoglobulin D switching can occur through homologous recombination in human B cells.Mol. Cell Biol. 1990; 10: 3690-3699Crossref PubMed Scopus (40) Google Scholar). In NHEJ, Ku quickly recognizes the broken DNA ends where it then functions as a platform to assemble other NHEJ factors including DNA-PKcs, Artemis, XLF, and DNA ligase IV/XRCC4. After minimal processing, the DNA ends are ligated via the DNA ligase IV/XRCC4 dimer (2Burma S. Chen B.P. Chen D.J. Role of non-homologous end joining (NHEJ) in maintaining genomic integrity.DNA Repair. 2006; 5: 1042-1048Crossref PubMed Scopus (310) Google Scholar). DNA double strand break non-homologous end-joining homologous recombination DNA-dependent protein kinase catalytic subunit exonuclease 1 Mre11/Rad50 Mre11/Rad50/Nbs1 Mre11/Rad50/Xrs2 yeast Ku forked DNA nucleotide(s). Although much work has been performed to identify and characterize factors that are required for repair by both DSB repair pathways, important questions are still unresolved, including what is the mechanism that modulates the pathway choice/switching between NHEJ and HR. Two factors are believed to play major roles in the choice of HR over NHEJ. First, the cell cycle phase is of importance as HR requires a homologous template; therefore, HR is believed to only be active during S and G2 phases of the cell cycle when a sister chromatid is available. NHEJ does not require a homologous template and is thus not restricted to a certain phase of the cell cycle but it is believed to be the dominant repair pathway in the G0 and G1 phases. The second factor is DNA end resection. The prevalent model for DNA end resection comes from genetic data generated from studies in Sacchromyces cerevisiae. The studies in yeast suggest a competition for DSB ends between the HR factors, Mre11 or Mre11/Rad50/Xrs2 (MRX), and the NHEJ factor, yeast Ku (yKu) (6Mimitou E.P. Symington L.S. Ku prevents Exo1- and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2.EMBO J. 2010; 29: 3358-3369Crossref PubMed Scopus (231) Google Scholar, 7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar). As both pathways require initial DNA damage sensing and processing, the choice between the two pathways may reside in which repair protein complex is initially assembled at DSB sites and how DNA ends are processed before ligation. Both MRX and yKu are recruited to DSB sites independently and simultaneously (8Wu D. Topper L.M. Wilson T.E. Recruitment and dissociation of nonhomologous end-joining proteins at a DNA double strand break in Saccharomyces cerevisiae.Genetics. 2008; 178: 1237-1249Crossref PubMed Scopus (99) Google Scholar) and lack of either complex results in an increase in the binding of the other complex and thus an increase in the other repair pathway (7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar, 8Wu D. Topper L.M. Wilson T.E. Recruitment and dissociation of nonhomologous end-joining proteins at a DNA double strand break in Saccharomyces cerevisiae.Genetics. 2008; 178: 1237-1249Crossref PubMed Scopus (99) Google Scholar). For example, in the absence of the MRX complex, yKu is accumulated at DSB sites and blocks Exo1-mediated DNA end resection (7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar). This data implicates that the MRX complex can mediate the dissociation of yKu from DSBs to allow DNA end resection to occur. The data in yeast suggests that competition and physical displacement of Ku from DNA ends via the MRX complex pushes the pathway choice to HR-mediated DSB repair instead of NHEJ. Although the studies in yeast produce a very compelling model for DSB repair pathway choice, the functional relationship between the HR and NHEJ pathways and the choice between the two may not directly apply to the human system. First, in yeast, the MRX complex is essential for classical NHEJ but it is not required for NHEJ in human cells and appears to only be involved in an alternative end-joining pathway. Second, Ku is very abundant (∼5 × 105 copies in HeLa cells) and NHEJ is more dominant in human cells, whereas yeast uses HR as its major DSB repair pathway (9Sonoda E. Hochegger H. Saberi A. Taniguchi Y. Takeda S. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair.DNA Repair. 2006; 5: 1021-1029Crossref PubMed Scopus (386) Google Scholar). For example, Ku deficiency causes severe radiosensitization in humans but Ku deficiency does not result in radiosensitivity in S. cerevisiae unless the HR protein Rad52 is also deleted (10Siede W. Friedl A.A. Dianova I. Eckardt-Schupp F. Friedberg E.C. The Saccharomyces cerevisiae Ku autoantigen homologue affects radiosensitivity only in the absence of homologous recombination.Genetics. 1996; 142: 91-102Crossref PubMed Google Scholar). Finally, there are structural differences between yKu and human Ku (11Aravind L. Koonin E.V. SAP, a putative DNA-binding motif involved in chromosomal organization.Trends Biochem. Sci. 2000; 25: 112-114Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar, 12Feldmann H. Driller L. Meier B. Mages G. Kellermann J. Winnacker E.L. HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae.J. Biol. Chem. 1996; 271: 27765-27769Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). As there are clear marked differences between the human and yeast Ku proteins, in this report we wanted to examine in vitro with purified human proteins whether Ku and Mre11 or Mre11/Rad50 compete with each other similarly as yKu and MRX for DNA ends and modulate DNA end resection. An in vitro assay system was established to biochemically study the interplay between human proteins in HR and NHEJ by using Ku, Mre11, Mre11/Rad50, and Exo1 and a specific DNA substrate with forked structure at one end. It was found that human Ku blocks Exo1-mediated resection and that neither Mre11 nor Mre11/Rad50 could actively mediate the displacement of Ku from the DNA end either physically or via its exonucleases or endonucleases activities to free the end for Exo1-mediated resection. Our data is in contrast with previous in vitro data generated with purified yeast proteins and suggest that the displacement of human Ku from DNA ends is more complicated than the direct competition with the MRX complex, which was established in yeast. The forked dsDNA was synthesized with a long single strand of 5′-CGCGCCCAGCTTTCCCAGCTAATAAACTAAAAACTCCTAAGG-3′ (42 nt) and a short single strand of 5′-CCTTAGGAGTTTTTAGTTTATTGGGCGCG-3′ (29 nt) (Invitrogen). The underlined nucleotides represented the hairpin sequence. 5′-End labeling of the long strand of the forked DNA was performed by incubating 2 μl of long strand of the forked DNA (10 μm), 1 μl of T4 polynucleotide kinase (10,000 units/ml), 5 μl of [γ-32P]ATP (3000 Ci/mmol, 10 mCi/ml), and 1 μl of cold ATP (200 μm) in a 20-μl reaction system at 37 °C for 30 min and stopped by heat inactivation at 65 °C for 20 min. 3′-End labeling of the short strand of the forked DNA was performed in a 50-μl reaction system containing 5 μl of short strand of the forked DNA (10 μm), 1 μl of terminal transferase (400 units/reaction), 5 μl of 3′-labeled [α-32P]Cordycepin 5′-triphosphate (5000 Ci/mmol, 10 mCi/ml) (Roche Applied Science), and 5 μl of CoCl2 (25 mm) at 37 °C for 15 min and stopped by addition of 5 μl of 0.2 m EDTA (pH 8.0). Both 5′- and 3′-labeled DNA were applied to a MicroSpin G-25 column to remove free 32P-nucleotide (GE Healthcare). The radiolabeled strand was annealed with the same molar amount of its complementary strand by incubating them together at 95 °C and cooled down to room temperature slowly. Labeling efficiency and DNA quality were examined using a 16% TBE native gel electrophoresis. DNA binding of Ku was performed using 10 nm forked DNA in a reaction system containing 25 mm Tris-HCl (pH 7.9), 50 mm KCl, 2 mm MgCl2, 1 mm EDTA, and 5% glycerol. Binding of Mre11 or Mre11/Rad50 to DNA in the absence or presence of Ku was performed using forked DNA in a binding buffer containing 25 mm MOPS (pH 7.0), 50 mm KCl, 1 mm DTT, and 1 mm MnCl2. All reactions were performed in a 10-μl system at room temperature for 15 min and 2 μl of loading dye (0.25% bromphenol blue, 0.25% xylene cyanol FF, and 30% glycerol) was added. The samples were separated via a 4% Tris glycine native gel, dried, and analyzed by Typhoon 9410 (GE Healthcare). All nuclease assays were performed in a 10-μl reaction. Exo1-mediated DNA resection was performed using 3′-end labeled forked DNA. To study the effect of Ku on Exo1-mediated DNA resection, 10 nm Exo1 and increasing concentrations of Ku (as indicated in Fig. 1B) were incubated with 3′-end labeled forked DNA in a reaction buffer containing 20 mm Hepes (pH 8.0), 50 mm KCl, 5 mm MgCl2, and 1 mm DTT at 37 °C for 1 h. Mre11-mediated DNA digestion was performed using 5′-end labeled forked DNA in a reaction buffer containing 25 mm MOPS (pH 7.0), 50 mm KCl, 1 mm DTT, and 1 mm MnCl2 at 37 °C for 1 h. To study the effect of Ku, increasing concentrations of Ku were added to the above reaction buffer. To differentiate nuclease activities of Mre11, 12 nm Mre11 was used for exonuclease activity and 60 nm Mre11 was used for endonuclease activity. To study whether the Mre11 helped Exo1 to process DNA in the presence of Ku, increasing concentrations of Ku were incubated with 10 nm Exo1, 120 nm Mre11, and 10 nm 5′-end labeled forked DNA in a reaction buffer containing 20 mm Hepes (pH 8.0), 50 mm KCl, 5 mm MgCl2, 0.5 mm MnCl2, and 1 mm DTT at 37 °C for 1 h. Mre11/Rad50-mediated exonuclease activity was performed in the same reaction buffer as for Mre11. For endonuclease activity of Mre11/Rad50, ATP (1 mm) was added to the same reaction buffer. To study the effect of Ku, increasing concentrations of Ku were added to the above reaction buffer. The effect of Mre11/Rad50 on Exo1-mediated DNA resection in the presence of Ku was examined in a two-step procedure. Increasing concentrations of Ku and 50 nm Mre11/Rad50 were incubated together with 10 nm 5′-end labeled forked DNA with or without ATP (1 mm) in a 10-μl reaction buffer containing 25 mm MOPS (pH 7.0), 40 mm KCl, 1 mm DTT, and 0.5 mm MnCl2 at 37 °C for 1 h. In the second step, 10 nm Exo1 and 5 mm MgCl2 were added to the reaction to a final volume of 12 μl and incubated for another 1 h at 37 °C. All reactions were stopped by adding an equal volume of formamide dye (95% formamide, 0.05% bromphenol blue, 0.05% xylene cyanol FF, 18 mm EDTA, 0.025% SDS), boiled at 95 °C for 5 min, and cooled down on ice for 5 min. The samples were separated by 16% urea-PAGE, dried, and analyzed in Typhoon 9410. Mre11 siRNAs were purchased from Thermo Fisher Scientific. The sequence for siRNA oligonucleotides against Mre11 was ACAGGAGAAGAGAUCAACUdTdT. Transfection of siRNA oligonucleotides was performed using Lipofectamine RNAi MAX (Invitrogen) according to the manufacturer's procedures. Live cell imaging combined with laser microirradiation was carried out as described previously with modifications (13Uematsu N. Weterings E. Yano K. Morotomi-Yano K. Jakob B. Taucher-Scholz G. Mari P.O. van Gent D.C. Chen B.P. Chen D.J. Autophosphorylation of DNA-PKcs regulates its dynamics at DNA double strand breaks.J. Cell Biol. 2007; 177: 219-229Crossref PubMed Scopus (312) Google Scholar, 14So S. Davis A.J. Chen D.J. Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites.J. Cell Biol. 2009; 187: 977-990Crossref PubMed Scopus (148) Google Scholar). Fluorescence was monitored by using an Axiovert 200M microscope (Carl Zeiss, Inc.), with a Plan Apochromat ×63/NA 1.40 oil immersion objective (Carl Zeiss, Inc.). A 365-nm pulsed nitrogen laser (Spectra Physics) was directly coupled to the epifluorescence path of the microscope and used to generate DSBs in a defined area of the nucleus. For quantitative analyses, standardized microirradiation conditions (minimal laser output of 75% for 5 pulses) were used to generate the same amount of DNA damage in each experiment. Time-lapse images were taken by an AxioCamHRm camera and the fluorescence intensities of microirradiated and nonirradiated areas within the cell nucleus were determined using the Axiovison Software, version 4.8 (Carl Zeiss, Inc.). To eliminate the influence of nuclear background fluorescence, the fluorescence intensity of an undamaged site in the same nuclei was subtracted from the fluorescence intensity of the accumulation spot for every cell at each time point. Nonspecific photobleaching and UV lamp output fluctuation were compensated for by correcting the accumulation site fluorescence intensity (IN) of each time point based on pre-laser background intensity using the formula: IN(t) = Idt/Ibt × IbpreIR, where Idt represents the difference between the accumulation spot intensity and the undamaged site background intensity of each time point, Ibt represents the background intensity of each time point, and IbpreIR represents the background intensity before irradiation. Relative fluorescence intensity (RF) was calculated using the formula: RF(t) = (INt − INpreIR)/(INmax − INpreIR), where INpreIR means IN of the microirradiated area before laser damage and INmax is the maximum IN in the microirradiated area of all time points. Each data point is the average of at least 10 independent measurements. Previous data generated in yeast showed that Ku may block DNA ends and that the MRX complex may actively displace Ku for DNA ends resection to occur. To study the mechanism of human Ku dissociation from the DNA end and determine whether it is similar to the yeast system, we first established in vitro binding conditions of human Ku70/80 to a model dsDNA substrate. The model dsDNA substrate used in the study is a forked DNA (F-DNA) substrate, which contains a 22-bp linear region connected to a branched structure that contains a 7-bp linear DNA on one end and a stem loop on the other (Fig. 1A). This forked DNA substrate was chosen because it allows Ku binding at the 22-bp linear region in only one direction and prevents its translocation off the DNA via the branched structure. The forked DNA structure with a 22-bp linear region also mimics the average 20-bp linker DNA found in nucleosomes, which likely only allows one directional loading of protein onto DNA in vivo. In addition, as 14 nt are required for Ku70/80 binding to DNA, the length of the linear region (22 bp) limits the binding of only one Ku to the model DNA substrate and allows the generation of a homogenous protein-DNA complex (15Walker J.R. Corpina R.A. Goldberg J. Structure of the Ku heterodimer bound to DNA and its implications for double strand break repair.Nature. 2001; 412: 607-614Crossref PubMed Scopus (880) Google Scholar). Finally, a similar structured DNA was used previously to solve the x-ray crystal structure of Ku70/80 as it assured uniform complex formation of Ku and DNA (16Yoo S. Kimzey A. Dynan W.S. Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end.J. Biol. Chem. 1999; 274: 20034-20039Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). First, quantitative binding of Ku to the model DNA substrate was performed by incubating increasing concentrations of Ku with 10 nm 32P-labeled forked DNA. One Ku (5–20 nm) and DNA band was observed in the 4% native Tris glycine gel representing 1:1 stoichiometry (Fig. 1B, lanes 2–4). The band intensities of both free DNA and Ku-bound DNA from the same binding reaction were measured to calculate their relative ratio. The values of ratios were expressed as percentage of total amount of DNA, which included free DNA and Ku-bound DNA and plotted as a histogram (Fig. 1C). It was found that 5 nm Ku was able to bind ∼80% of the model forked DNA, whereas 10 to 20 nm Ku occupied nearly 100% of the model DNA (Fig. 1C). It should be noted that at higher concentrations of Ku70/80 a small amount of higher order Ku-DNA species were observed (Fig. 1B and data not shown). These are likely due to the ability of Ku to unstably bind to shorter stretches of DNA, but the vast majority of the Ku-DNA species observed were 1 Ku with 1 DNA (15Walker J.R. Corpina R.A. Goldberg J. Structure of the Ku heterodimer bound to DNA and its implications for double strand break repair.Nature. 2001; 412: 607-614Crossref PubMed Scopus (880) Google Scholar). In summary, a stable 1:1 stoichiometric binding was formed between 5 and 20 nm Ku and 10 nm forked DNA and the open end of the forked DNA substrate is clearly bound by Ku. As yKu has been implicated in blocking Exo1-mediated end resection in the absence of the MRX complex, we wanted to establish an in vitro DNA end resection system with human proteins to test if human Ku could block end resection by human Exo1 using the defined DNA substrate (7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar). Recombinant human Exo1 and Ku70/80 were purified from Sf9 insect cells following expression via recombinant baculoviruses (supplemental Fig. S1A and data not shown). First, the exonuclease activity of Exo1 was studied by incubating various concentrations of Exo1 (1–10 nm) with the forked DNA substrate, which was radiolabeled at the 3′-end at 37 °C for 1 h. After the end of the reaction, DNA was denatured and separated by a 16% urea denaturing gel. The efficiency of DNA resection was determined by observing reduction of the original DNA substrate and appearance of resected DNA fragments. Low concentration of Exo1 (1 nm) resected DNA modestly as it produced an intermediate DNA fragment of ∼15 nt as marked by an arrow (Fig. 2A, lane 2). More thorough DNA resection was observed with increased concentrations of Exo1 as the amount of the original DNA was nearly lost and two newly resected DNA fragments were observed as marked by a bracket (Fig. 2A, lanes 3 and 4). To examine whether human Ku blocks human Exo1-mediated DNA resection, DNA was incubated with 10 nm Exo1 and increasing concentrations of Ku. Only very limited Exo1 resection was observed in the reaction when 5 nm Ku was added to the reaction (Fig. 2B, lane 3). As shown in Fig. 1, B and C, addition of 5 nm Ku resulted in ∼80% forked DNA bound with Ku and ∼20% unbound. Thus the limited resection observed in the presence of 5 nm Ku70/80 is likely due to Exo1-mediated resection of the 20% unbound DNA. When the Ku:forked DNA ratio was at a 1:1 molar ratio (as measured in Fig. 1, B and C) Exo1-mediated resection of the model DNA substrate was completely abrogated. This data shows that similar to the data generated with yeast; human Ku70/80 can bind to DNA substrates and block Exo1-mediated DNA end resection. In S. cerevisiae, either Mre11 or Rad50 deficiency causes excess Ku accumulation at DSBs, which suggests that the MR and Ku complexes compete for the same DSB ends (7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar). In S. cerevisiae, Mre11 itself rather than its nuclease activity is required for DNA resection and repair of HO-induced DSBs suggesting that structurally Mre11 can regulate and compete for DNA ends (7Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (175) Google Scholar, 17Llorente B. Symington L.S. The Mre11 nuclease is not required for 5′ to 3′ resection at multiple HO-induced double strand breaks.Mol. Cell Biol. 2004; 24: 9682-9694Crossref PubMed Scopus (125) Google Scholar). Taking this into consideration, we next wanted to test the competition between human Ku and purified recombinant Mre11 or Mre11/Rad50 for DNA ends in vitro (supplemental Fig. S1, B and C). First, quantitative binding of Mre11 to the model DNA substrate was performed by incubating varying concentrations of Mre11 with the F-DNA. The gel shift data shows that at low concentrations, the Mre11-DNA complex produced smeared signals in the native gel (Fig. 3A, lanes 2 and 3). Increasing the concentration (120 and 250 nm) of Mre11 resulted in the formation of high order Mre11-DNA complexes (Fig. 3A). The data suggests that Mre11 has relatively low DNA binding affinity and likely forms heterogeneous complexes with the F-DNA substrate. To study the competition between Mre11 and Ku70/80 for binding to F-DNA in vitro, F-DNA was incubated with 20 nm Ku and either 12 or 60 nm Mre11 simultaneously. Because the Mre11-DNA and Ku-DNA complexes migrated differently in the native gel (Figs. 1B and 3A, respectively), direct competition between Ku and Mre11 for DNA binding can be determined by the disappearance of one of the protein-DNA signals. Ku-DNA formed the only major protein-DNA complex when 12 nm Mre11 was added to the reaction (Fig. 3B, lane 5). The Ku-DNA complex was the dominant protein-DNA complex when 60 nm Mre11 was added, suggesting that Mre11 could not compete with Ku for DNA binding (Fig. 3B, lane 6). However, a new band with higher molecular weight than the Ku-DNA complex was observed when 60 nm Mre11 and 20 nm Ku were incubated together (Fig. 3B, lane 6). Furthermore, the addition of 60 nm Mre11 appears to result in a switch in the dominant Ku-DNA complex from one Ku binding to the F-DNA to two Ku. This suggests that Mre11 and Ku may be able to simultaneously bind the same DNA substrate and possibly change the binding of Ku to DNA. Furthermore, the data suggests that Mre11 may interact with Ku itself. To test whether Ku and Mre11 can interact independent of DNA, we tested protein-protein interaction with purified proteins. After incubation of purified Ku and Mre11 at 4 °C, each protein was immunoprecipitated via a specific antibody and tested whether they interact in the absence of DNA in vitro. The results show that Ku and Mre11 fail to interact in vitro (supplemental Fig. S2). We conclude that Ku and Mre11 do not physically interact and Mre11 is not able to block Ku from binding to DNA or mediate its dissociation from DNA. Rad50 makes a complex with Mre11 and confers allosteric regulation on Mre11 by coordinating the binding of Mre11 to DNA. In addition, ATP hydrolysis mediated by the ABC ATPase of Rad50 allows an open to close conformation of Mre11/Rad50 (18Williams G.J. Williams R.S. Williams J.S. Moncalian G. Arvai A.S. Limbo O. Guenther G. SilDas S. Hammel M. Russell P. Tainer J.A. ABC ATPase signature helices in Rad50 link nucleotide state to Mre11 interface for DNA repair.Nat. Struct. Mol. Biol. 2011; 18: 423-431Crossref PubMed Scopus (131) Google Scholar). Hence, we repeated the gel shift assays with Mre11/Rad50 to determine whether DNA binding was different from Mre11 and if Mre11/Rad50 could outcompete Ku for or mediate the dissociation of Ku from DNA ends. The condition of the Mre11/Rad50 binding reaction was similar to Mre11 except ATP was also included in a set of reactions to test a potential effect of ATP hydrolysis in binding to the F-DNA substrate. Like Mre11, Mre11/Rad50 binding also showed smeared signals regardless of the presence of ATP (Fig. 4A). However, its binding affinity was much lower than Mre11 because most DNA was left unbound and the smeared signals were weak even with 250 nm Mre11/Rad50 (Fig. 4A, lanes 4 and 8). When the concentration of MR was increased, a number of sharp bands were observed, possibly representing different stoichiometry between Mre11/Rad50 and DNA (Fig. 4B, lanes 3, 4 and 7, 8). In addition, unlike Mre11 alone, a number of sharp bands were generated by the Mre11/Rad50-DNA complex at higher Mre11/Rad50 concentrations, which suggests that Mre11/Rad50-DNA complexes are more uniform than the Mre11-DNA complex. Data also showed that ATP hydrolysis may affect the DNA binding affinity of the Mre11/Rad50 complex as a slight reduction in DNA binding was observed with the inclusion of ATP (Fig. 4B). The potential competition between Mre11/Rad50 and Ku for DNA binding was studied by incubating increasing concentrations of Mre11/Rad50 (25–250 nm) and Ku (20 nm) together. As shown in Fig. 4C, even high concentrations of Mre11/Rad50 (250 nm) with or without ATP cannot compete with Ku for DNA ends. Furthermore, unlike Mre11, it appears that Mre11/Rad50 cannot bind to the same DNA substrate as Ku. Taken together, the data shows Mre11 has higher binding affinity to the forked DNA substrate than Mre11/Rad50 and Mre11 partially changed Ku-DNA binding. However, neither Mre11 nor Mre11/Rad50 was able to" @default.
• W2037004712 created "2016-06-24" @default.
• W2037004712 creator A5043686371 @default.
• W2037004712 creator A5052822702 @default.
• W2037004712 creator A5071731539 @default.
• W2037004712 creator A5081758694 @default.
• W2037004712 date "2012-02-01" @default.
• W2037004712 modified "2023-09-28" @default.
• W2037004712 title "Human Ku70/80 Protein Blocks Exonuclease 1-mediated DNA Resection in the Presence of Human Mre11 or Mre11/Rad50 Protein Complex" @default.
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