Calcium Stiffens Archaeal Rad51 Recombinase from Methanococcus voltae for Homologous Recombination

Archaeal RadA or Rad51 recombinases are close homologues of eukaryal Rad51 and DMC1. These and bacterial RecA orthologues play a key role in DNA repair by forming helical nucleoprotein filaments in which a hallmark strand exchange reaction between homologous DNA substrates occurs. Recent studies have discovered the stimulatory role by calcium on human and yeast recombinases. Here we report that the strand exchange activity but not the ATPase activity of an archaeal RadA/Rad51 recombinase from Methanococcus voltae (MvRadA) is also subject to calcium stimulation. Crystallized MvRadA filaments in the presence of CaCl2 resemble that of the recently reported ATPase active form in the presence of an activating dose of KCl. At the ATPase center, one Ca2+ ion takes the place of two K+ ions in the K+-bound form. The terminal phosphate of the nonhydrolyzable ATP analogue is in a staggered conformation in the Ca2+-bound form. In comparison, an eclipsed conformation was seen in the K+-bound form. Despite the changes in the ATPase center, both forms harbor largely ordered L2 regions in essentially identical conformations. These data suggest a unified stimulation mechanism by potassium and calcium because of the existence of a conserved ATPase center promiscuous in binding cations. Archaeal RadA or Rad51 recombinases are close homologues of eukaryal Rad51 and DMC1. These and bacterial RecA orthologues play a key role in DNA repair by forming helical nucleoprotein filaments in which a hallmark strand exchange reaction between homologous DNA substrates occurs. Recent studies have discovered the stimulatory role by calcium on human and yeast recombinases. Here we report that the strand exchange activity but not the ATPase activity of an archaeal RadA/Rad51 recombinase from Methanococcus voltae (MvRadA) is also subject to calcium stimulation. Crystallized MvRadA filaments in the presence of CaCl2 resemble that of the recently reported ATPase active form in the presence of an activating dose of KCl. At the ATPase center, one Ca2+ ion takes the place of two K+ ions in the K+-bound form. The terminal phosphate of the nonhydrolyzable ATP analogue is in a staggered conformation in the Ca2+-bound form. In comparison, an eclipsed conformation was seen in the K+-bound form. Despite the changes in the ATPase center, both forms harbor largely ordered L2 regions in essentially identical conformations. These data suggest a unified stimulation mechanism by potassium and calcium because of the existence of a conserved ATPase center promiscuous in binding cations. In homologous recombination, a hallmark DNA strand exchange reaction is promoted by RecA-like recombinases between a single-stranded DNA (ssDNA) and a homologous double-stranded DNA (dsDNA). Such a reaction plays critical role in the repair of double-stranded DNA breaks and restart of stalled replication forks (1Cox M.M. Genes Cells. 1998; 3: 65-78Crossref PubMed Scopus (104) Google Scholar, 2Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (875) Google Scholar, 3Courcelle J. Ganesan A.K. Hanawalt P.C. BioEssays. 2001; 23: 463-470Crossref PubMed Scopus (59) Google Scholar, 4Lusetti S.L. Cox M.M. Annu. Rev. Biochem. 2002; 71: 71-100Crossref PubMed Scopus (356) Google Scholar, 5Kowalczykowski S.C. Trends Biochem. Sci. 2000; 25: 156-165Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar). This recombinase superfamily (6Seitz E.M. Kowalczykowski S.C. Mol. Microbiol. 2000; 37: 555-560Crossref PubMed Scopus (29) Google Scholar) is composed of bacterial RecA (7Clark A.J. Margulies A.D. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 451-459Crossref PubMed Scopus (467) Google Scholar), archaeal RadA or Rad51 (8Sandler S.J. Satin L.H. Samra H.S. Clark A.J. Nucleic Acids Res. 1996; 24: 2125-2132Crossref PubMed Scopus (104) Google Scholar), and eukaryal Rad51 (9Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar), and meiosis-specific DMC1 (10Bishop D.K. Park D. Xu L. Kleckner N. Cell. 1992; 69: 439-456Abstract Full Text PDF PubMed Scopus (980) Google Scholar). Large differences in their primary structures exist between bacterial RecAs and nonbacterial orthologues. Their tertiary and quaternary structures, however, are clearly conserved (11VanLoock M.S. Yu X. Yang S. Lai A.L. Low C. Campbell M.J. Egelman E.H. Structure (Camb.). 2003; 11: 187-196Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 12Conway A.B. Lynch T.W. Zhang Y. Fortin G.S. Fung C.W. Symington L.S. Rice P.A. Nat. Struct. Mol. Biol. 2004; 11: 791-796Crossref PubMed Scopus (241) Google Scholar, 13Wu Y. He Y. Moya I.A. Qian X. Luo Y. Mol. Cell. 2004; 15: 423-435Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Such filamentous assemblies are classic allosteric systems equipped with at least two functional sites, one located at the subunit interface for binding and hydrolyzing ATP and the other located near the filament axis for binding DNA and promoting strand exchange. In vitro studies have clearly shown that optimal reaction conditions differ for the well characterized Escherichia coli RecA (EcRecA) and members of the archaeal/eukaryal group (14Rice K.P. Eggler A.L. Sung P. Cox M.M. J. Biol. Chem. 2001; 276: 38570-38581Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Liu Y. Stasiak A.Z. Masson J.Y. McIlwraith M.J. Stasiak A. West S.C. J. Mol. Biol. 2004; 337: 817-827Crossref PubMed Scopus (48) Google Scholar, 16Sehorn M.G. Sigurdsson S. Bussen W. Unger V.M. Sung P. Nature. 2004; 429: 433-437Crossref PubMed Scopus (151) Google Scholar). EcRecA favors low salt conditions with little or no monovalent cation. Human and yeast Rad51 and DMC1, on the other hand, favor the presence of a salt, typically KCl or (NH4)2SO4. Fishel and coworkers further found that human Rad51 is stimulated by K+ or bigger monovalent cations (17Shim K.S. Schmutte C. Yoder K. Fishel R. DNA Repair. 2006; 5: 718-730Crossref PubMed Scopus (27) Google Scholar). Using MvRadA 2The abbreviations used are: MvRadA, RadA recombinase from M. voltae; EcRecA, RecA recombinase from E. coli; ATPγS, adenosine 5′-O-(thiotriphosphate); AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. as a prototype, we have structurally rationalized that monovalent cations stimulate RadA/Rad51/DMC1 by bridging and stabilizing a critical L2 region with the ATP cofactor (18Wu Y. Qian X. He Y. Moya I.A. Luo Y. J. Biol. Chem. 2005; 280: 722-728Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Qian X. He Y. Wu Y. Luo Y. J. Mol. Biol. 2006; 360: 537-547Crossref PubMed Scopus (18) Google Scholar). However, the reported MvRadA structures could not explain the recently discovered stimulatory role by divalent Ca2+ ions on human Rad51 (20Bugreev D.V. Mazin A.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9988-9993Crossref PubMed Scopus (196) Google Scholar) and DMC1 (21Bugreev D.V. Golub E.I. Stasiak A.Z. Stasiak A. Mazin A.V. J. Biol. Chem. 2005; 280: 26886-26895Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Though initially suspected of being relevant only to higher eukaryotes in sensing the second messenger Ca2+, similar effects have later been found for yeast DMC1 (22Lee M.H. Chang Y.C. Hong E.L. Grubb J. Chang C.S. Bishop D.K. Wang T.F. J. Biol. Chem. 2005; 49: 40980-40984Abstract Full Text Full Text PDF Scopus (43) Google Scholar). To test whether a similar effect by Ca2+ exists in archaeal orthologues, we carried out assays on MvRadA in the presence of Ca2+. The strand exchange activity but not the ATPase activity of MvRadA is stimulated by Ca2+, suggesting that this archaeal orthologue also possesses a structural element for sensing Ca2+. We further determined the crystal structure of MvRadA in the presence of Ca2+. A largely ordered L2 is anchored by Ca2+ with the ATP cofactor in an essentially identical conformation to that previously elucidated in the K+-activated forms of MvRadA. Analysis of sequence and structure suggests that archaeal/eukaryal recombinases may share a negatively charged cavity lined by ATP, the catalytic Glu, an Asp in the ATP cap, and the C terminus of a short helix in the L2 region. We interpret these results as suggesting a unified stimulation mechanism by various cations because of the existence of such a conserved and promiscuous cation-binding pocket in the ATPase center of closely related RadA/Rad51/DMC1 recombinases. Cloning, Protein Preparation, and Crystallization—RadA from Methanococcus voltae was subcloned into pET28a (Novagen). The recombinant proteins were overexpressed in BL21-CodonPlus (DE3)-RIPL cells (Stratagene) and purified as reported (13Wu Y. He Y. Moya I.A. Qian X. Luo Y. Mol. Cell. 2004; 15: 423-435Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 19Qian X. He Y. Wu Y. Luo Y. J. Mol. Biol. 2006; 360: 537-547Crossref PubMed Scopus (18) Google Scholar). In brief, the purification procedure involved steps of polymin P (Sigma) precipitation, high salt extraction, and three chromatography steps using heparin (Amersham Biosciences), hydroxyapatite (Bio-Rad), and DE52 (Whatman) columns. The RadA protein pooled between 0.3 and 0.4 m NaCl from the DE52 column was concentrated to ∼30 mg/ml by ultra-filtration. Strand Exchange Assay Using Virion DNA—Circular single-stranded ϕX174 virion DNA (5386 nucleotides) and its homologous double-stranded ϕX174 replication form I DNA (5386 base pairs) were purchased from New England Biolabs. The double-stranded replication form I DNA was linearized by PstI (VWR) digestion before being used as the dsDNA substrate. The reaction solutions were composed of 50 mm Tris-Hepes buffer at pH 7.4, specified amount of CaCl2 or MgCl2, 5 mm ATP or AMP-PNP. The reaction solution with ATP was supplemented with an ATP regenerating system composed of 4 mm creatine phosphate and 0.01 unit/μl creatine The single-stranded ϕX174 virion DNA or in nucleotides) and MvRadA were in the reaction buffer at for single-stranded protein from E. coli (VWR) was to a of a second at for the dsDNA was to a of in base a at for of and of were to the reaction A further for was to MvRadA. A was with 5 of buffer composed of and and a The DNA was for 4 in a of 4 The was with a Strand exchange were using the Strand exchange were by the of the by the of the dsDNA in Strand Exchange Assay Using and a homologous double-stranded DNA were used as the strand exchange substrates as previously X. He Y. Wu Y. Luo Y. J. Mol. Biol. 2006; 360: 537-547Crossref PubMed Scopus (18) Google Scholar). The solution for strand exchange reaction was composed of 5 mm ATP or an mm or m 50 mm buffer at pH 7.4, MvRadA, and The was at with MvRadA for before the dsDNA substrate. The reaction was at by to a of mm and to a of of digestion at a was with 5 of buffer composed of and and a The was with and with the and Structure concentrated MvRadA protein was using the at a of The optimal well solution for mm AMP-PNP, m MgCl2, m m and m buffer at pH to a of mm and 0.4 mm in 4 and were purchased from The other used in were from were to solution composed of the solution supplemented with and for to in a by an The were and using a system as (13Wu Y. He Y. Moya I.A. Qian X. Luo Y. Mol. Cell. 2004; 15: 423-435Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). crystal was by into and data at the at the A of of and were This data was using W. J. Scopus Google Scholar). The previously MvRadA was used as the for The was using J. Struct. Biol. PubMed Scopus Google Scholar) and using P. J. M. Rice T. Biol. 1998; PubMed Scopus Google Scholar). Using the data with the a was using by ordered calcium near the ATP analogue was located at a in the to in and Using the data the calcium to a higher in the and also to higher to of the and are in The were using P. J. 24: Google Scholar) and using D.J. J. Mol. Google Scholar). The and structure of the data have been in the Protein data and structure in to in the is is structure with The is using a of the the in to in the is is The is using a of the the in a Ca2+ Strand Exchange of have recently rationalized the on MvRadA by potassium ions (18Wu Y. Qian X. He Y. Moya I.A. Luo Y. J. Biol. Chem. 2005; 280: 722-728Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Qian X. He Y. Wu Y. Luo Y. J. Mol. Biol. 2006; 360: 537-547Crossref PubMed Scopus (18) Google Scholar). K+ ions an ATPase and strand conformation by in a negatively charged cavity at the ATPase However, potassium and other monovalent ions are not the only cations to To out whether a similar effect by divalent Ca2+ ions exists in archaeal orthologues as reported for human and yeast Rad51 and DMC1 recombinases (20Bugreev D.V. Mazin A.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9988-9993Crossref PubMed Scopus (196) Google Scholar, D.V. Golub E.I. Stasiak A.Z. Stasiak A. Mazin A.V. J. Biol. Chem. 2005; 280: 26886-26895Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, M.H. Chang Y.C. Hong E.L. Grubb J. Chang C.S. Bishop D.K. Wang T.F. J. Biol. Chem. 2005; 49: 40980-40984Abstract Full Text Full Text PDF Scopus (43) Google Scholar), we carried out ATPase and strand exchange assays on MvRadA in the presence of divalent cations Ca2+ and Despite a of monovalent cations (18Wu Y. Qian X. He Y. Moya I.A. Luo Y. J. Biol. Chem. 2005; 280: 722-728Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and divalent cations and Ca2+, data not K+ the only that the ATPase activity of MvRadA. In comparison, other monovalent and divalent cations of the ATPase activity in the presence of mm KCl. The stimulation on the strand exchange activity of MvRadA, on the other hand, to the presence of ions such as but not to to its on human and yeast Ca2+ the strand exchange activity of MvRadA ATPase activity not In the presence of 5 mm ATP, of mm CaCl2 was for of the A of was in the presence of 4 mm is that the was in the of the only previously for this We also to DNA the strand exchange between which not the presence of single-stranded by Ca2+ was in the presence of ATP or that stimulation by Ca2+ on DNA strand exchange is a by RadA/Rad51/DMC1 recombinases. for a structural of the mechanism of calcium we carried out strand exchange in the presence of AMP-PNP, the only ATP analogue to MvRadA In the presence of 5 mm and mm MvRadA strand exchange between The were similar to in the presence of We MvRadA in the presence of and was found to an in the of could in its The strand exchange activity in the presence of and of and CaCl2 was before to of conditions and structural The structure was to a of using an data calcium is the element in the the was used to a Ca2+ near the of A data of was later at the at the which in and with the of a Ca2+ in this and studies of MvRadA to group with similar helical and essentially identical The filament with the axis is previously reported forms using the and The Ca2+-bound structure is and similar to the previously determined K+-bound form of MvRadA (18Wu Y. Qian X. He Y. Moya I.A. Luo Y. J. Biol. Chem. 2005; 280: 722-728Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, X. Wu Y. He Y. Luo Y. 2005; PubMed Scopus Google Scholar). The Ca2+-bound form also an ion in a seen in and The of and the from this to its the seen in other crystal AMP-PNP, the nonhydrolyzable ATP analogue is between MvRadA subunit the ATP analogue and the largely its conserved to M. A. Trends Biochem. Sci. 15: Full Text PDF PubMed Scopus Google Scholar) and the The subunit the ATP to and the of the L2 region in The L2 region to an helix to to helix of EcRecA Nature. 1992; PubMed Scopus Google Scholar). The of forms a with the of the ATP Ca2+ ion located by forms a between the and three at the C terminus of the the of this Ca2+ form a The of and one of the ATP analogue the and two in and the The Ca2+ ion with or two of its three A for the hydrolyzing is with the of and from the subunit the The of EcRecA and have been as the catalytic on the EcRecA crystal structure Nature. 1992; PubMed Scopus Google Scholar) and to J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), for a the the group of and the the effects by and the Ca2+ the is further for the by the and by and However, the terminal phosphate is in a staggered conformation which may the in the eclipsed conformation seen in the K+-bound ATPase active form X. Wu Y. He Y. Luo Y. 2005; PubMed Scopus Google Scholar). This may explain calcium not stimulate optimal ATPase The L2 which is for strand exchange is in an essentially identical conformation as seen in the K+-bound form. forms for a from to In comparison, in the L2 region are in the form of MvRadA in the presence of of or in the of an amount of a stimulatory cation. both potassium and calcium ions are elucidated to a largely ordered L2 which is with the of the ions in DNA strand we these as further for the that a L2 region is critical for promoting DNA strand exchange as by Mol. Microbiol. 2005; PubMed Scopus Google center of MvRadA in The is from that in by MvRadA are in and Ca2+ and K+ ions are in and ions and are and The is in a Ca2+-bound form. previously reported K+-bound form The L2 regions are in essentially identical in both Ca2+ and K+ are in a conserved cavity lined by the of the the catalytic in the ATP cap, and the C terminus of a short helix in the L2 region. The terminal phosphate is in a staggered conformation in the Ca2+-bound form but in an eclipsed conformation in the K+-bound Large Strand Exchange of structural between and Ca2+-bound structures a promiscuous cavity of and of cations. the of the Ca2+ ion in the crystal form between the two K+ ions in the previously reported form We further the that this cavity could the a Ca2+, 5 mm was to DNA strand exchange to of the In comparison, a CaCl2 was to of its stimulatory effect is that the We that the binding of or other ions in the structurally characterized cavity the of the of the cavity by the of In the presence of 5 mm ATP, an and Ca2+ were by using the C. S. D. 2004; PubMed Scopus Google Scholar). mm is a of mm is to MvRadA. A Ca2+ of mm to the dose of mm We that one or two ions could in the MvRadA cavity but with Ca2+. In the presence of AMP-PNP, not to stimulate MvRadA in promoting strand exchange between DNA or is not that previously reported MvRadA structures in the presence of not a second ion in with the of AMP-PNP. The archaeal RadA protein from M. voltae is a RecA orthologue that the hallmark strand exchange reaction between homologous with its sequence with eukaryal the archaeal RadA from M. voltae is by K+ and Ca2+ MvRadA as a structural for the mechanism of its human active recombinase DNA filament is in a strand which an and form of DNA as with E.H. J. Mol. Biol. 2001; PubMed Scopus Google Scholar). The of an active filament also the binding of and ATP at the ATPase center of which the because of the ATPase activity ATP is or ATP is by exchange. on the crystal structure of E. coli two located and L2 have been as the structural for binding DNA Nature. 1992; PubMed Scopus Google Scholar). This been by as Rev. Biochem. Mol. Biol. 2003; PubMed Scopus Google Scholar). In the crystal structures of MvRadA, two to and L2 of EcRecA are to the filament axis seen in the EcRecA structure (13Wu Y. He Y. Moya I.A. Qian X. Luo Y. Mol. Cell. 2004; 15: 423-435Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Their for binding DNA. RecA-like recombinases are allosteric proteins with a between its ATPase center and The stimulatory of potassium ions on the ATPase and strand exchange of MvRadA (18Wu Y. Qian X. He Y. Moya I.A. Luo Y. J. Biol. Chem. 2005; 280: 722-728Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Qian X. He Y. Wu Y. Luo Y. J. Mol. Biol. 2006; 360: 537-547Crossref PubMed Scopus (18) Google Scholar) to the that changes in its ATPase center and L2 region the L2 and its determined in the presence of strand exchange ions K+ and Ca2+ harbor largely ordered L2 regions in a conformation as with a one in structures determined in the of ions or in the presence of X. Wu Y. He Y. Luo Y. 2005; PubMed Scopus Google Scholar). These are not only with the that L2 is critical for promoting DNA strand exchange but also a to ATP and and are A between the ATP cofactor and the short helix in the L2 region in the of L2 as well as in further ATP for The negatively charged of the cation-binding pocket is further by two conserved one from the Asp in the ATP and the other from the catalytic the short helix its C terminus this such a L2 the and Ca2+-bound structures a for the on archaeal and eukaryal recombinases. that MvRadA and its eukaryal homologues as well is of both monovalent and divalent cations of various The of helical of the Ca2+-bound MvRadA filament with the K+-bound one to a between the ions are This the 0.3 of Ca2+. The is by one of the Ca2+ to further the M. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The ATP in with the ions also from the eclipsed conformation in the K+-bound form to the staggered conformation in the Ca2+-bound form. Though with both and are the stabilizing effects by these two conserved are in forming with the of Ca2+. This of the ATPase center for promiscuous binding of cations stabilizing a L2 for promoting with DNA substrates and In the of a stimulatory or in the of ATP L2 X. Wu Y. He Y. Luo Y. 2005; PubMed Scopus Google Scholar). This of in similar ATPase such as the in the bacterial protein D. N. S. A. Nat. Struct. Mol. Biol. 2006; PubMed Scopus Google Scholar). because of the changes in the ATPase center, the ATP, by one Ca2+ or two may in its for Though crystal structures of MvRadA to suggest a unified mechanism by monovalent and divalent cations on this archaeal In of the that such archaeal and eukaryal recombinases share a high of in with their in not that share this conserved structural element for In as out by and Mazin (20Bugreev D.V. Mazin A.V. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9988-9993Crossref PubMed Scopus (196) Google Scholar), sensing Ca2+ as a second messenger could play a role on recombinase activity in higher Though we that the primary Ca2+, which the L2 is to play a role in the is to two calcium ions located by is in with the L2 The second Ca2+ is by the and of the AMP-PNP, the of and the of In a previously reported filament in which L2 is largely this cavity is by a potassium of a at this not to with the in the L2 region. cations such as potassium and calcium may play a role in the of the of the The Ca2+ an seen in previously reported MvRadA This is by We a MvRadA, Y. and Y. the crystal structure of which no a near However, this protein was to in promoting DNA strand exchange. is calcium binding near play a role in activating MvRadA by the in the L2 region. In of in eukaryal of DNA strand exchange with K+ or from to active nucleoprotein filaments M.G. Sigurdsson S. Bussen W. Unger V.M. Sung P. Nature. 2004; 429: 433-437Crossref PubMed Scopus (151) Google Scholar, M.H. Chang Y.C. Hong E.L. Grubb J. Chang C.S. Bishop D.K. Wang T.F. J. Biol. Chem. 2005; 49: 40980-40984Abstract Full Text Full Text PDF Scopus (43) Google Scholar, D. M. T. J. C. R. C. Nucleic Acids Res. 2005; PubMed Scopus Google Scholar). the other hand, two have been in the crystal structures of MvRadA. In the presence of or in the of the L2 region is suggesting that this form an X. Wu Y. He Y. Luo Y. 2005; PubMed Scopus Google Scholar). The of this form that its L2 region is not in with DNA. of into nucleoprotein filaments is The filaments in the presence of and as well as calcium or high of potassium harbor a largely ordered L2 region and may resemble the active filament previously seen by The ordered L2 and regions form a in this active form that their stabilizing with DNA are optimal for the to Archaeal RadA and eukaryal proteins share a conserved by the this ions such as K+ or Ca2+ this conformation and for DNA and of active nucleoprotein We and for with the at the We also of the