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Heteroduplex Joint Formation Free of Net Topological Change by Mhr1, a Mitochondrial Recombinase

2009; Elsevier BV; Volume: 284; Issue: 14 Linguagem: Inglês

10.1074/jbc.m900023200

1083-351X

Feng Ling, Minoru Yoshida, Takehiko Shibata,

DNA and Nucleic Acid Chemistry

Homologous pairing, an essential process for homologous recombination, is the formation of a heteroduplex joint by an invading single-stranded DNA tail and a complementary sequence within double-stranded DNA (dsDNA). The base rotation of the parental dsDNA, to switch from parental base pairs to heteroduplex ones with the invading single-stranded DNA, sterically requires vertical extension between adjacent base pairs, which inevitably induces untwisting of the dsDNA. RecA is a prototype of the RecA/Rad51/Dmc1 family proteins, which promote ATP-dependent homologous pairing in homologous DNA recombination in vivo, except in mitochondria. As predicted by the requirement for the untwisting, dsDNA bound to RecA is extended and untwisted, and homologous pairing by RecA in vitro is extensively stimulated by the negative supercoils of dsDNA substrates. D-loop formation in negatively supercoiled dsDNA, which serves as an assay for homologous pairing, is also catalyzed in an ATP-independent manner by proteins structurally unrelated to RecA, such as Mhr1. Mhr1 is required for yeast mitochondrial DNA recombination instead of RecA family proteins. Inconsistent with the topological requirements, tests for the effects of negative supercoils revealed that Mhr1 catalyzes homologous pairing with relaxed closed circular dsDNA much more efficiently than with negatively supercoiled dsDNA. Topological analyses indicated that neither the process nor the products of homologous pairing by Mhr1 involve a net topological change of closed circular dsDNA. This would be favorable for homologous recombination in mitochondria, where dsDNA is unlikely to be under topological stress toward unwinding. We propose a novel topological mechanism wherein Mhr1 induces untwisting without net topological change. Homologous pairing, an essential process for homologous recombination, is the formation of a heteroduplex joint by an invading single-stranded DNA tail and a complementary sequence within double-stranded DNA (dsDNA). The base rotation of the parental dsDNA, to switch from parental base pairs to heteroduplex ones with the invading single-stranded DNA, sterically requires vertical extension between adjacent base pairs, which inevitably induces untwisting of the dsDNA. RecA is a prototype of the RecA/Rad51/Dmc1 family proteins, which promote ATP-dependent homologous pairing in homologous DNA recombination in vivo, except in mitochondria. As predicted by the requirement for the untwisting, dsDNA bound to RecA is extended and untwisted, and homologous pairing by RecA in vitro is extensively stimulated by the negative supercoils of dsDNA substrates. D-loop formation in negatively supercoiled dsDNA, which serves as an assay for homologous pairing, is also catalyzed in an ATP-independent manner by proteins structurally unrelated to RecA, such as Mhr1. Mhr1 is required for yeast mitochondrial DNA recombination instead of RecA family proteins. Inconsistent with the topological requirements, tests for the effects of negative supercoils revealed that Mhr1 catalyzes homologous pairing with relaxed closed circular dsDNA much more efficiently than with negatively supercoiled dsDNA. Topological analyses indicated that neither the process nor the products of homologous pairing by Mhr1 involve a net topological change of closed circular dsDNA. This would be favorable for homologous recombination in mitochondria, where dsDNA is unlikely to be under topological stress toward unwinding. We propose a novel topological mechanism wherein Mhr1 induces untwisting without net topological change. Homologous DNA recombination plays critical roles in the repair of double-stranded DNA (dsDNA) 3The abbreviations used are: dsDNA, double-stranded DNA; mtDNA, mitochondrial DNA; ssDNA, single-stranded DNA; cc-dsDNA, closed circular dsDNA. breaks and meiotic disjunctions of homologous chromosomes in the nuclear genome (for reviews, see Refs. 1O'Driscoll M. Jeggo P.A. Nat. Rev. Genet. 2006; 7: 45-54Crossref PubMed Scopus (463) Google Scholar and 2Neale M.J. Keeney S. Nature. 2006; 442: 153-158Crossref PubMed Scopus (296) Google Scholar) and in DNA replication as well as DNA repair in yeast mitochondria (3Ling F. Morioka H. Ohtsuka E. Shibata T. Nucleic Acids Res. 2000; 28: 4956-4963Crossref PubMed Google Scholar) (see Ref. 4Shibata T. Ling F. Mitochondrion. 2007; 7: 17-23Crossref PubMed Scopus (35) Google Scholar for review). A key intermediate step in homologous recombination is homologous pairing, in which a single-stranded DNA (ssDNA) tail derived from a double-stranded break invades a homologous sequence within intact dsDNA, resulting in heteroduplex joint formation with the complementary sequence of the dsDNA by replacing a parental strand. Heteroduplex joints are formed by switching base pairs of the parental dsDNA to heteroduplex base pairs between the invading ssDNA and the complementary sequence of the dsDNA, which requires extension between adjacent base pairs of the dsDNA to provide a space for the rotation of bases for the base pair switch (5Nishinaka T. Shinohara A. Ito Y. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11071-11076Crossref PubMed Scopus (71) Google Scholar). The extension of dsDNA is necessarily associated with its untwisting (6Stasiak A. DiCapua E. Koller T. J. Mol. Biol. 1981; 151: 557-564Crossref PubMed Scopus (144) Google Scholar, 7Stasiak A. DiCapua E. Nature. 1982; 299: 185-186Crossref PubMed Scopus (168) Google Scholar), which generates positive supercoiling in the remaining region in closed circular dsDNA (cc-dsDNA; Fig. 1, from DNA 1 to DNA 2). A negatively supercoiled cc-dsDNA substrate neutralizes the positive supercoils, but a relaxed cc-dsDNA substrate accumulates the positive supercoils, which will resist the formation of heteroduplex joints. Homologous pairing activities are typically assayed in vitro by examining the formation of D-loops, using negatively supercoiled cc-dsDNA (natural cc-dsDNA, called "form I") and homologous ssDNA oligonucleotides as substrates (8Shibata T. DasGupta C. Cunningham R.P. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1638-1642Crossref PubMed Scopus (279) Google Scholar, 9Beattie K.L. Wiegand R.C. Radding C.M. J. Mol. Biol. 1977; 116: 783-803Crossref PubMed Scopus (88) Google Scholar). A D-loop consists of a heteroduplex joint and a loop of a displaced parental strand of the dsDNA, with the associated untwisting of the parental dsDNA (9Beattie K.L. Wiegand R.C. Radding C.M. J. Mol. Biol. 1977; 116: 783-803Crossref PubMed Scopus (88) Google Scholar, 10Holloman W.K. Wiegand R. Hoessli C. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2394-2398Crossref PubMed Scopus (71) Google Scholar). Thus, the formation of a D-loop in cc-dsDNA by itself also generates positive supercoils. Consistent with these topological requirements in homologous pairing and D-loop formation, negative supercoils were shown to be essential for uncatalyzed D-loop formation (10Holloman W.K. Wiegand R. Hoessli C. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 2394-2398Crossref PubMed Scopus (71) Google Scholar) and to stimulate RecA- and Rad51-catalyzed D-loop formation extensively, as compared with the initial velocity with dsDNA without supercoils (11Shibata T. DasGupta C. Cunningham R.P. Williams J.G.K. Osber L. Radding C.M. J. Biol. Chem. 1981; 256: 7565-7572Abstract Full Text PDF PubMed Google Scholar, 12Van Komen S. Petukhova G. Sigurdsson S. Stratton S. Sung P. Mol. Cell. 2000; 6: 563-572Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). RecA is the prototype of the RecA/Rad51/Dmc1 family proteins, which are essential to catalyze homologous pairing through an ATP-dependent reaction in homologous DNA recombination in prokaryotic genomes and eukaryotic nuclei (8Shibata T. DasGupta C. Cunningham R.P. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1638-1642Crossref PubMed Scopus (279) Google Scholar, 13McEntee K. Weinstock G.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 2615-2619Crossref PubMed Scopus (241) Google Scholar, 14Shinohara A. Ogawa H. Matsuda Y. Ushio N. Ikeo K. Ogawa T. Nat. Genet. 1993; 4: 239-243Crossref PubMed Scopus (30) Google Scholar, 15Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (755) Google Scholar, 16Bishop D.K. Park D. Xu L.Z. Kleckner N. Cell. 1992; 69: 439-456Abstract Full Text PDF PubMed Scopus (980) Google Scholar, 17Li Z. Golub E.I. Gupta R. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11221-11226Crossref PubMed Scopus (128) Google Scholar). Cellular DNA is likely to be relaxed by the potent activities of DNA topoisomerases, but the factors required to fulfill the topological requirement for homologous pairing in vivo remain unidentified, especially in mitochondria. Homologous recombination in bacterial viruses and the mitochondria of yeasts and mammals is independent of the RecA/Rad51/Dmc1 family proteins. Instead, we found that in yeast, mtDNA recombination depends on Mhr1, a protein structurally unrelated to RecA/Rad51 (3Ling F. Morioka H. Ohtsuka E. Shibata T. Nucleic Acids Res. 2000; 28: 4956-4963Crossref PubMed Google Scholar, 18Ling F. Makishima F. Morishima N. Shibata T. EMBO J. 1995; 14: 4090-4101Crossref PubMed Scopus (74) Google Scholar) and that Mhr1-catalyzes in vitro D-loop formation in an ATP-independent manner (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Mitochondrial DNA (mtDNA) encodes essential components of the machinery that drives oxidative respiration-dependent energy production. mtDNA undergoes extensive mitotic homologous recombination in the yeast Saccharomyces cerevisiae (20Dujon B. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1981: 505-635Google Scholar) and humans (21Kraytsberg Y. Schwartz M. Brown T.A. Ebralidse K. Kunz W.S. Clayton D.A. Vissing J. Khrapko K. Science. 2004; 304: 981Crossref PubMed Scopus (213) Google Scholar, 22Zsurka G. Hampel K.G. Kudina T. Kornblum C. Kraytsberg Y. Elger C.E. Khrapko K. Kunz W.S. Am. J. Hum. Genet. 2007; 80: 298-305Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In addition, Mhr1 is a critical player in the vegetative segregation of heteroalleles that leads to a genetic state in which all of the copies of mtDNA in each cell or in each individual (in the case of healthy human babies) share an identical sequence (called "homoplasmy") (20Dujon B. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1981: 505-635Google Scholar). The function of Mhr1 in homoplasmy is in sharp contrast to the generally accepted roles of homologous recombination in nuclear genomes; i.e. homologous recombination, and in particular meiotic recombination, contributes to genetic diversification. Mhr1 functions in homoplasmy to initiate the rolling circle mode of mtDNA replication to form concatemers, which are selectively transmitted to daughter cells, leading to homoplasmy (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar, 23Ling F. Shibata T. Mol. Biol. Cell. 2004; 15: 310-322Crossref PubMed Scopus (52) Google Scholar). RecA does not initiate this mode of DNA replication in wild type Escherichia coli cells (24Cohen A. Clark A.J. J. Bacteriol. 1986; 167: 327-335Crossref PubMed Google Scholar). Thus, it is important to identify the differences and similarities between the homologous pairing catalyzed by the RecA/Rad51/Dmc1 family proteins and that catalyzed by Mhr1 to provide insights into the differences between the two genetic outcomes. The ATP-independent D-loop formation mediated by Mhr1 and other proteins structurally unrelated to RecA may be a type of complementary strand annealing (i.e. a two-strand reaction) through the mechanism proposed for uncatalyzed D-loop formation. This idea is based on the following observations. Negatively supercoiled dsDNA tends to melt and create ssDNA regions (9Beattie K.L. Wiegand R.C. Radding C.M. J. Mol. Biol. 1977; 116: 783-803Crossref PubMed Scopus (88) Google Scholar), and some non-RecA proteins (λ phage β protein, RecT, Rad52, and RecO) have potent activity to anneal complementary ssDNA molecules (25Muniyappa K. Radding M.C. J. Biol. Chem. 1986; 261: 7472-7478Abstract Full Text PDF PubMed Google Scholar, 26Hall S.D. Kane M.F. Kolodner R.D. J. Bacteriol. 1993; 175: 277-287Crossref PubMed Google Scholar, 27Mortensen U.H. Bendixen C. Sunjevaric I. Rothstein R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10729-10734Crossref PubMed Scopus (388) Google Scholar, 28Sugiyama T. New J.H. Kowalczykowski S.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6049-6054Crossref PubMed Scopus (261) Google Scholar). If this model is correct, then Mhr1 would absolutely require negative supercoiling for the apparently homologous pairing. Alternatively, the D-loop formation catalyzed by non-RecA proteins is a true homologous pairing reaction, including ssDNA invading an internal, homologous sequence in dsDNA (i.e. a three-strand reaction). This model was proposed based on structural analysis of an ssDNA oligonucleotide bound to either RecA or Rad51 in the presence of a nonhydrolyzable ATP analogue, which suggested that homologous pairing is an intrinsic function of DNA molecules rather than a specific function of proteins (5Nishinaka T. Shinohara A. Ito Y. Yokoyama S. Shibata T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11071-11076Crossref PubMed Scopus (71) Google Scholar, 29Shibata T. Nishinaka T. Mikawa T. Aihara H. Kurumizaka H. Yokoyama S. Ito Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8425-8432Crossref PubMed Scopus (58) Google Scholar). In this study, topological analyses originally aimed at testing the above alternative models revealed an unexpected feature of the Mhr1-catalyzed homologous pairing, which is prevented by negative supercoils and generates no net topological changes of dsDNA substrates during and after the reactions. This finding suggests a novel mechanism wherein Mhr1 extends and untwists dsDNA to promote homologous pairing. Purification of Mhr1-The purified Mhr1 used in this study was obtained as previously described (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Standard Reaction Buffer-The standard buffer consisted of 50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 10 mm MgCl2, and 1 mm dithiothreitol. DNA Substrates-The methods for the preparation of supercoiled pGsat4 and fX174 RF I (replicative form I) cc-dsDNA and ssDNA oligonucleotide (50-mer oligonucleotides) and for the 5′-end labeling of the ssDNA oligonucleotide with 32P were described previously (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Negatively supercoiled pUC18 plasmid DNA was prepared from pUC18 plasmid DNA-harboring E. coli XL2-blue cells (endA1 gyrA96(nalR) thi-1 recA1 relA1 lac glnV44 F′ [::Tn10 proAB+ lacIq Δ(lacZ)M15] hsdR17(rK- mK+)) (Stratagene), using an illustra plasmidPrep Mini Spin Kit (GE Healthcare). The amounts of DNA are expressed as the amounts of nucleotides, unless otherwise stated. Preparation of Relaxed cc-dsDNA-Partially or fully relaxed cc-dsDNA was prepared by treating negatively supercoiled pUC18 plasmid DNA (209 μm in nucleotides) with 0.025 units/μl or 0.1 units/μl calf thymus topoisomerase I (Takara Shuzo Co., Kyoto, Japan) in a 20-μl reaction mixture at 37 °C for 30 min. The topoisomerase I was then completely removed from the reaction solution by a phenol/chloroform extraction, and the DNA was purified with an illustra plasmidPrep Mini Spin Kit. When we analyzed the Mhr1-catalyzed formation of three-stranded structures in the presence of active topoisomerase I, which should release any topological stress during the reaction, the topoisomerase removal and DNA purification procedures were not performed. Two-dimensional gel electrophoresis was performed as described previously to analyze the relaxed cc-dsDNA (30Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar, 31Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar). Standard D-loop Formation Assay-The standard reaction mixture (20.5 μl) for the assay consisted of 15.6 mm Tris-HCl (pH 7.5), 1.8 mm dithiothreitol, 88 μg/ml bovine serum albumin, and 1 mm MgCl2. After the 32P-labeled ssDNA oligonucleotide (1.0 μm final concentration after dsDNA was added, unless otherwise stated) was incubated with 1.95 μm (final concentration) Mhr1 in the reaction mixture (19.5 μl) for 5 min at 37 °C, 1 μl of dsDNA was added to a concentration of 15 μm, and the mixture was incubated at 37 °C for the indicated period of time. For the reaction with E. coli RecA, 1.3 mm ATP and MgCl2 (final concentration, 13 mm) were added together with the dsDNA. After the proteins were removed, the products (D-loops) were assayed by agarose gel electrophoresis. After electrophoresis, the gel was dried and exposed to an imaging plate, which was analyzed with a Fuji BAS2000 image analyzer. Details of the assay have been described previously (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). A Restriction Endonuclease Protection Assay for Homologous Pairing-The homologous three-stranded structures, formed as a result of homologous pairing, are protected from restriction enzymes. A restriction endonuclease protection assay based on this principle was performed according to a previously described method (32Pezza R.J. Voloshin O.N. Vanevski F. Camerini-Otero R.D. Genes Dev. 2007; 21: 1758-1766Crossref PubMed Scopus (86) Google Scholar), with a slight modification. In four sets of the reaction mixture (7.0 μl), Mhr1 at various concentrations was incubated with an ssDNA oligonucleotide (1.3 μm final concentration after dsDNA was added) bearing the sequence encompassing either the NdeI site from pUC18 (NdeI ssDNA oligonucleotide, 5′-CGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGAT-3′; in two sets) or the SspI site from pUC18 (SspI ssDNA oligonucleotide, 5′-AATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT-3′; in two sets) in the standard reaction buffer at 37 °C for 30 min. After the incubation, to each sample containing the Mhr1-ssDNA oligonucleotide complexes (7.0 μl), 3.0 μl of supercoiled or relaxed pUC18 dsDNA or ScaI-linearized pUC18 dsDNA (to a final concentration of 41.8 μm) were added, and the mixture was incubated at 37 °C for 30 min. To each one from the two sets of reaction mixtures containing the same ssDNA oligonucleotide, the NdeI or SspI restriction endonuclease (1.0 unit/μl or 0.5 units/μl, respectively) was then added, and the samples were incubated at 37 °C for 30 min to allow DNA cleavage. When the formation of homologous three-stranded structures by Mhr1 was analyzed in the absence of topological stress (i.e. in the presence of eukaryotic topoisomerase I, which relaxes both negative and positive supercoils), we first relaxed the samples of negatively supercoiled pUC18 cc-dsDNA (209 μm) with 0.025 units/μl or 1.0 unit/μl calf thymus topoisomerase I at 37 °C for 30 min in a 20-μl reaction mixture. After a 1.5-fold dilution of the reaction solution containing the relaxed pUC18 cc-dsDNA (final concentration, 41.8 μm) and active topoisomerase I (final concentration, 0.005 or 0.2 units/μl) by the standard reaction buffer, 3 μl of the diluted dsDNA containing the topoisomerase were added to the Mhr1-ssDNA oligonucleotide complexes (7.0 μl) and mixed, and the mixture was incubated at 37 °C for 30 min. The NdeI was then added, and the samples were incubated at 37 °C for 30 min. After the DNA substrates were incubated with Mhr1 and then treated with a restriction enzyme, all of the proteins were removed by the addition of 1 μl of 10% SDS and 1 μl of 10 mg/ml proteinase K, followed by an incubation at 37 °C for 15 min. The DNA was then analyzed by electrophoresis on 1% agarose gels in the presence of 0.3 μg/ml ethidium bromide. The signals from the various DNA species in the gel were quantified by a Southern blot analysis, using [32P] pUC18 DNA as a probe, after the DNA was transferred to N+ nylon membranes. The signals were analyzed with a Fuji BAS2000 image analyzer (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Electrophoretic Assay for the Protein-free Products of Homologous Pairing by Mhr1 in the Presence of Topoisomerase I Using cc-dsDNA Substrates-In each reaction (20 μl), negatively supercoiled cc-dsDNA (pUC18 plasmid DNA (245 μm) or φX174 RF I cc-dsDNA (172 μm)) was treated in the standard reaction buffer with 1.0 unit/μl calf thymus topoisomerase I at 37 °C for 30 min. Then 3.0 μl of relaxed cc-dsDNA (final concentration in the reaction mixture, 49.0 μm for pUC18 plasmid DNA and 34.5 μm for φX174 RF I cc-dsDNA) in standard buffer containing active topoisomerase were added to 7.0 μl of a solution containing 32P-labeled ssDNA oligonucleotide (1.3 μm calculated final concentration, assuming that all DNA was recovered during the labeling and purifying processes) and 0 or 3.9 μm (final concentration) Mhr1 in the standard reaction buffer, and the reaction mixture was incubated at 37 °C for 30 min in the presence of 0.2 units/μl topoisomerase I. After the proteins were removed by adding 1 μl of 10% SDS and 1 μl of 10 mg/ml proteinase K at 37 °C for 20 min, the DNA products were analyzed by electrophoresis on 1% agarose gels (2 V/cm) for 11 h in a cold room (4 °C). The products were also assayed by two-dimensional electrophoresis on 1% agarose gels in the cold room, as described (30Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar, 31Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar). After electrophoresis, the gel was dried and exposed to an imaging plate, which was analyzed with a Fuji BAS2000 image analyzer, as described previously (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Assay for the Topological Status of the Products of Homologous Pairing by Mhr1-Relaxed cc-dsDNA was prepared by incubating negatively supercoiled pUC18 plasmid DNA (209 μm) with 1.0 unit/μl calf thymus topoisomerase I at 37 °C for 30 min. To initiate Mhr1-catalyzed homologous pairing, the relaxed pUC18 cc-dsDNA and active calf thymus topoisomerase I were added to the reaction mixture (the final dsDNA concentration and the amount of the topoisomerase I were ∼41.8 μm and 0.2 units/μl, respectively) containing Mhr1-NdeI ssDNA oligonucleotide complexes, formed by the incubation of 1.3 μm NdeI-ssDNA oligonucleotides with 3.9 μm Mhr1. The reaction mixture was incubated at 37 °C for 30 min. As a control, Mhr1 was omitted from the reaction. After a phenol/chloroform extraction, the DNA products were alkaline-treated (pH 13.0) to dissociate the ssDNA oligonucleotide from the cc-dsDNA, neutralized to reform the double helix, and then analyzed by two-dimensional electrophoresis on 1% agarose gels. The first and second dimensions of gel electrophoresis were both performed at 1.3 V/cm for 11 h at room temperature, but the second one was performed in the presence of 0.02 μg/ml ethidium bromide at room temperature, as described (30Kikuchi A. Asai K. Nature. 1984; 309: 677-681Crossref PubMed Scopus (194) Google Scholar, 31Nakasu S. Kikuchi A. EMBO J. 1985; 4: 2705-2710Crossref PubMed Google Scholar). Assay for cc-dsDNA Binding by Mhr1-In each reaction (10 μl), negatively supercoiled cc-dsDNA (pUC18 plasmid DNA, 41.8 μm in nucleotides) was mixed with Mhr1 at various concentrations (0, 0.93, and 1.9 μm) in the standard reaction buffer and incubated at 37 °C for 30 min. The Mhr1-cc-dsDNA complexes were then subjected to a gel shift assay, as previously described (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Assay for Topological Changes of cc-dsDNA Due to Mhr1 Binding-In each reaction (20 μl), negatively supercoiled cc-dsDNA (pUC18 plasmid DNA; 209 μm) was treated with 1.0 unit/μl calf thymus topoisomerase I at 37 °C for 30 min. Then the reaction mixture was diluted 1.5-fold by the standard reaction buffer, 3 μl of the diluted solution (containing relaxed cc-dsDNA, final concentration 41.8 μm) were added to 7.0 μl of a solution containing Mhr1 at various concentrations (0, 0.93, and 1.9 μm), and the solution was incubated at 37 °C for 30 min in the presence of 0.2 units/μl topoisomerase I. The DNA products were analyzed by electrophoresis on 1% agarose gels (2 V/cm) for 11 h at room temperature, after the topoisomerase I was removed by a phenol/chloroform extraction. Mhr1 Promotes D-loop Formation in Vitro from Negatively Supercoiled cc-dsDNA and Homologous ssDNA Oligonucleotides-When negatively supercoiled cc-dsDNA and homologous ssDNA oligonucleotide fragments (or ssDNA oligonucleotides) are incubated in the presence of an excess amount of an ATP-dependent homologous pairing protein (RecA or Dmc1), D-loops are quickly formed and then are actively dissociated in an ATP-hydrolysis-dependent manner. This process occurs as a part of "the D-loop cycle" (34Shibata T. Ohtani T. Iwabuchi M. Ando T. J. Biol. Chem. 1982; 257: 13981-13986Abstract Full Text PDF PubMed Google Scholar, 35Wu A.M. Kahn R. DasGupta C. Radding C.M. Cell. 1982; 30: 37-44Abstract Full Text PDF PubMed Scopus (48) Google Scholar, 36Enomoto R. Kinebuchi T. Sato M. Yagi H. Shibata T. Kurumizaka H. Yokoyama S. J. Biol. Chem. 2004; 279: 35263-35272Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). We found that purified Mhr1 promotes D-loop formation from these substrates via a reaction that is independent of ATP (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). Unlike the RecA/Dmc1-catalyzed reaction, in the Mhr1-catalyzed reaction, the D-loops were formed as a result of a simple time-dependent reaction (Fig. 2). D-loop formation depended on the presence of Mhr1 and the homology between the dsDNA and ssDNA oligonucleotide (Fig. 2). After the proteins were removed, the products of the Mhr1-promoted reaction were determined to be authentic D-loops, because they migrated on gels with the same velocity as that observed for D-loops formed by RecA, and they dissociated following the cleavage of the dsDNA outside the homologous region (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar). This is critical proof for D-loop formation by Mhr1, since it is well established that in the absence of a topological constraint toward untwisting (generated by negative supercoils of dsDNA substrate), D-loops spontaneously and quickly dissociate (37Radding C.M. Beattie K.L. Holloman W.K. Wiegand R.C. J. Mol. Biol. 1977; 116: 825-839Crossref PubMed Scopus (92) Google Scholar). We next studied the effects of negative supercoils and topological constraints of cc-dsDNA substrates on Mhr1-catalyzed homologous pairing. Due to the characteristics described above, the D-loop formation assay is not suitable to detect the products of homologous pairing using dsDNA substrates with variable topological conditions. Therefore, we quantified the Mhr1-catalyzed homologous pairing activity using the restriction endonuclease protection assay described by Ferrin and Camerini-Otero (38Ferrin L.J. Camerini-Otero R.D. Science. 1991; 254: 1494-1497Crossref PubMed Scopus (184) Google Scholar) as a reliable assay for homologous pairing, to test the effects of supercoils and topological constraints. This assay is based on the principle that the three-stranded structures formed through homologous pairing of dsDNA and a ssDNA oligonucleotide by homologous DNA-pairing proteins, such as RecA, are resistant to restriction endonucleases (38Ferrin L.J. Camerini-Otero R.D. Science. 1991; 254: 1494-1497Crossref PubMed Scopus (184) Google Scholar). To detect the formation of homologous three-stranded structures, we first used negatively supercoiled plasmid cc-dsDNA (pUC18) bearing a single NdeI cleavage site and a single SspI site as the dsDNA substrate and ssDNA oligonucleotides containing a sequence homologous to the region around the NdeI (NdeI ssDNA oligonucleotide) or SspI (SspI ssDNA oligonucleotide) cleavage site. If three-stranded structures formed as a result of homologous pairing after the incubation of the dsDNA with the NdeI or SspI ssDNA oligonucleotide and a homologous pairing protein, then the dsDNA would become resistant to NdeI or SspI, respectively, whereas it would remain sensitive to SspI or NdeI, respectively (Fig. 3A, a). The resistance was dependent on the incubation with Mhr1 and the combination of the oligonucleotide and the restriction endonuclease that recognized the restriction site within the oligonucleotide (Fig. 3B, a). For optimal protection of the dsDNA by Mhr1, the ssDNA oligonucleotide and Mhr1 had to be incubated together before the addition of dsDNA; the simultaneous addition of the ssDNA oligonucleotide and dsDNA decreased the degree of protection, whereas the incubation of the dsDNA with Mhr1 before the addition of the ssDNA oligonucleotide did not protect the dsDNA from the endonuclease (data not shown). The signals representing the protected dsDNA with a three-stranded structure became smaller when an excess amount of Mhr1 was added (Fig. 3, B (a and c) and C (a and c)). This and previous results (19Ling F. Shibata T. EMBO J. 2002; 21: 4730-4740Crossref PubMed Scopus (79) Google Scholar) showed that excessive levels of the enzyme prevent Mhr1-catalyzed homologous pairing of dsDNA and the ssDNA oligonucleotide, as also observed with two other AT