DNA Strand Exchange Promoted by RecA K72R

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C.(1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg•dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg•dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5′-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some are in the of a in a of DNA strand exchange produces a DNA by a in the are The mutant protein also the of the but at a Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C.(1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg•dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg•dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5′-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some are in the of a in a of DNA strand exchange produces a DNA by a in the are The mutant protein also the of the but at a RecA protein of is a with a of The protein is in and is to the of DNA of the to DNA and the of at and and and RecA protein promotes a of DNA strand exchange that in and DNA The or four DNA RecA a nucleoprotein the or DNA DNA is with a duplex strand the a of hybrid is to the a strand exchange to to the DNA the of the to the RecA The reaction also a of structural barriers in the DNA protein is a with a of to wild type RecA DNA protein of ATP is the nucleoprotein When a duplex DNA is to the the to and at that the DNA strand exchange reaction as as ATP is and and The reaction is with of hybrid DNA The of ATP is by the that RecA protein DNA strand exchange some in the presence of ATP that is not by RecA and by the RecA mutant K72R and Kowalczykowski, and with wild type RecA protein in the presence of and The in the K72R mutant in a and the mutant protein binds but does not hydrolyze ATP. The mutant also a DNA strand with and Kowalczykowski, that the RecA to at DNA and DNA strand exchange ATP hydrolysis, and to for ATP in DNA strand The RecA to the hybrid DNA of a DNA strand exchange reaction does RecA protein hydrolyze to the of RecA-mediated ATP is to the limitations of that of that the strand exchange is in and bidirectional and The also not structural barriers in the DNA and and not four DNA ATP to to the DNA strand exchange required in for that barriers and for exchange some but the with of RecA-mediated DNA strand exchange in the absence of ATP ATP and strand exchange to with exchange is a formation of a of hybrid DNA product at the reaction or the is of exchange is that the reaction does not to The or of strand exchange that in some of strand exchange The to with ATP to RecA and the and Kowalczykowski, and the in a DNA that as a strand exchange and in ATP is of a duplex DNA to the of ATP by to The observed is to the length of in the that the length of is a or with over of or and strand exchange RecA the DNA or to the same to a of strand exchange in some length of hybrid DNA is by a in the hybrid DNA is The is as in hybrid DNA formation ATP is and and in the ATP is not is to the to strand exchange is the of the the of the length of the the of the and reaction and present a that the is in The also of the reaction and to with or of ATP. The a of with RecA K72R reaction and the RecA K72R mutant of the DNA strand exchange reaction promoted by the RecA K72R mutant protein but the to of the the of the RecA and RecA protein and as The RecA protein concentration by at of and DNA protein as with the that a to of DNA The concentration of protein by at of and a of by the of The S. and and and a and and and and duplex DNA and is with a the the of is with the into the and duplex DNA and DNA and as and The concentration of and by at and as DNA concentrations are in of duplex DNA by of DNA by The protein by with and by duplex DNA by DNA with and or as The with by with and The by duplex DNA a RecA The reaction DNA with protein, and ATP. strand exchange for The reaction by and to and The at for The reaction with and and the DNA in a The reaction in a at The duplex DNA as The duplex is and the K72R K72R by a a DNA the of the reaction with the for at 72 and the the into The of the by and the the with RecA in to The of the K72R by the K72R a of and with in for at with by at at in a The with and The in to of The in and at of the RecA K72R RecA K72R protein by a of the for the wild type RecA of and as by The the by to The in of and of the same to of a The with and the of the RecA protein in the by in the by to by in of and of to a 7 The with of and with of to by and of to of to a protein for a and the with of and with of to and of in and at The of the RecA K72R protein The RecA K72R protein at as by a of a The concentration of the RecA K72R protein the same as wild type RecA The RecA K72R protein of or at in a strand exchange reaction and ATP or a duplex DNA and or duplex DNA with RecA protein or RecA K72R protein for the concentrations of and protein to the in some as The and as as the of of RecA K72R and in some as in the and the with for at and for at the over The of DNA by the a and the with to for in the the to the of the strand exchange reaction as the of the DNA in a some the with to the concentration of Mg2+ in of that in a with The concentration of Mg2+ the of for the of the at and the by the of of in a at some of the with as and or of the RecA K72R and the wild type RecA protein by a and to the and at for to the of the of to the of ATP or also and RecA protein as in strand exchange reaction by the of protein and ATP or to concentrations of and for electron microscopy by the strand exchange reaction with to by electron microscopy to migration by of to a concentration of at for and with for at The with the The with and for at The into and for at type and as and and of the DNA molecules as and of the electron microscopy to that the reaction observed the molecules are for some and in the to the of the duplex that in DNA strand exchange to by the and duplex DNA molecules in a DNA substrate molecules and that to or levels of reaction in the in some the by and in a by to the length of hybrid DNA for a of in some of the of of of in of the of exchange as the of to duplex DNA into pairs of DNA the length of the The into of exchange and as of exchange with the DNA as a substrate in some in into of into of in and as The four of by the at the the for the not The also by the to the same that is in the with the at and ATP and wild type RecA or RecA K72R The not the or as a at by of of at for by The and the of the electron in are for or a at the of is a of the DNA strand exchange reaction promoted by RecA K72R as a to to and strand exchange to that mutant protein the by and the for RecA DNA strand exchange in as a to in the the of the K72R mutant protein to strand of structural barriers in the DNA strand and DNA strand exchange with four DNA to in We also the of the mutant protein to the of RecA K72R ATP or a the of the mutant and wild type with ATP and The mutant not a of ATP or of the for ATP and to and The is and the with the in observed by and RecA K72R protein also for at and with observed in The for the protein to in the presence of and for ATP and hydrolysis, with RecA K72R DNA in the of strand exchange DNA and in the presence of ATP and reaction by the presence of DNA strand exchange reaction observed with RecA K72R the same reaction the wild type RecA protein promoted reaction When as the the mutant protein of the substrate DNA to strand exchange strand exchange a product in some with the DNA at The reaction of the mutant protein in the reaction observed in the presence of the wild type RecA reaction of the DNA into completed hybrid DNA The are in with the by and with the RecA K72R mutant strand exchange promoted by RecA as and with or RecA K72R and with also ATP or duplex DNA as a and and the are and duplex DNA the are and to the duplex product of DNA strand the duplex the and reaction of DNA by RecA K72R DNA by the concentration in DNA strand exchange with the in the presence of a of joint molecules is observed with Mg2+ as Mg2+ concentrations the of joint molecules but a to strand exchange duplex DNA molecules with over 7 of hybrid DNA The to at Mg2+ concentrations to the concentration, of the Mg2+ in a with the the product formation to concentration, the at a of The with is in hybrid DNA but a concentration of Mg2+ of are Mg2+ in and concentration a of hybrid DNA the concentration of Mg2+ required to in with the concentration The of joint strand exchange also with concentration, levels of Mg2+ required for optimal of The also as a of the concentration of Mg2+ in of that in the Mg•dATP and the of hybrid DNA is at a Mg2+ is The of strand exchange a of Mg2+ is DNA strand exchange is affected by the concentration of for as and and RecA K72R protein, and with The and as and and show with concentrations at and and Mg2+ concentrations at the of the are as in and show the of as a of Mg2+ concentration, with hybrid DNA in and in and the same are as a of Mg2+ concentration to that in a Mg•dATP as and The concentrations in The and a for the of DNA strand that the K72R mutant protein a strand exchange reaction over 7 of hybrid DNA at Mg2+ that in the Mg•dATP is required for optimal uptake of substrate duplex DNA into the nucleoprotein to form strand exchange but the same Mg2+ the formation of the of hybrid DNA to the completed The uptake of substrate duplex DNA Mg2+ concentrations are to the of optimal for in the generation of strand exchange by the K72R mutant is nucleoprotein and over optimal Mg2+ the concentrations of and Mg2+ are of the duplex DNA to in The of is at and by and the of the nucleoprotein When levels of Mg2+ the generation of greatly over a the presence of at but not at or with The generation of at and to at Mg2+ the at of hybrid DNA by RecA K72R is as in with RecA K72R and with also and show the with and are and to are as in The and DNA as in is a of the product formation for the in and the formation of joint in of in and for Mg2+ concentrations in and to the length of hybrid DNA that by the RecA K72R mutant protein in the presence of not The RecA K72R with a duplex reaction with a DNA some product formation in the but the reaction that promoted by the When the of as the duplex product generation not in product formation promoted by RecA K72R with DNAs the We that the mutant protein the generation of over of hybrid with hybrid DNA observed at as a of the and Mg2+ concentrations and of to to of the K72R mutant protein the of strand exchange and also the formation of strand exchange not with the mutant protein are observed is to the The generation of strand exchange optimal also required a concentration of the RecA K72R mutant protein to the The generation of not at the concentration of mutant protein the of of not protein, or in the does not the the that Mg2+ formation of but subsequent migration to in strand exchange at Mg2+ concentration, and by the of Mg2+ or The concentration at in of Mg2+ to optimal for product formation in the Mg2+ to the migration to to a the not the of product When the reaction at as to the Mg2+ concentration to that of the product formation The reaction the in not as as that observed in with indicating that the of Mg2+ produces a of The same observed to Mg2+ not The of Mg2+ a at the reaction product formation to that observed in Mg2+ not The that the concentration of Mg2+ that of are that a that is not Mg2+ concentrations are of hybrid DNA or by as and RecA K72R protein, and with also are the of or to the at and the Mg2+ concentration in the reaction the to The reaction are and to and the the a reaction in the reaction the and and the reaction into into a and a and or RecA K72R, and DNA in The for 7 with the in and to are as in The and DNA as in DNA duplex DNA with of or the to strand to a to the strand exchange reaction promoted by RecA of DNA that are to the When ATP is protein promotes DNA strand exchange to to the strand of the duplex that is to the the to the of the duplex DNA substrate as and the a DNA strand exchange reaction and with and protein, the duplex DNA with into a as a in strand exchange to the of hybrid DNA When is to the the reaction is and of hybrid DNA are RecA K72R strand exchange the the or with in the with to to or the Mg2+ concentration to or the of with the Mg2+ concentrations not strand exchange is bidirectional with RecA as and with or RecA K72R and The DNA with or also are and are as in with the and are the RecA with and a reaction and by electron microscopy as and of DNA in by RecA K72R, a and DNA in by electron and the of hybrid DNA in The and that the reaction with RecA K72R with substantial of the duplex and of hybrid We that strand exchange by the K72R mutant is DNA RecA DNA formation of hybrid DNA by the mutant to to and Kowalczykowski, to a strand exchange a The duplex DNA substrate a DNA with a the and of The substrate does not the or the and is to the duplex substrate The product of a strand exchange reaction is a DNA with a duplex and a over DNA strand exchange does not structural barriers in the duplex DNA as and or RecA K72R or and of the duplex substrate the reaction the the and are DNA in are DNA with The reaction for four are and to The reaction DNA molecules by a strand duplex as a the RecA K72R the reaction of the duplex DNA with reaction that with but product formation observed for the reaction a in the duplex DNA RecA DNA strand in the the generation of with the K72R and reaction the at the of the of the in the presence of protein and of the product the duplex the The Mg2+ concentration the of the mutant protein to the not a of with Mg2+ concentrations to in but completed with the mutant protein by electron microscopy at the the RecA but completed in duplex or duplex of with as the type and and into a in strand exchange to the The molecules in the observed at levels in of is in the of a for DNA the of duplex or duplex molecules in the product form and 7 with microscopy of DNA in a structural in the duplex into as in reaction with and as and and show reaction in the reaction with RecA the of are of the and a reaction product in the reaction with a of reaction in the in strand exchange to of the duplex molecules observed in the and RecA K72R is the RecA K72R of DNA in by RecA K72R, a and of strand exchange also for the reaction by RecA K72R and in the of the duplex and the of hybrid in strand exchange to the to by the of or DNA molecules present in DNA We that RecA K72R protein not of DNA strand RecA K72R a strand exchange with the K72R mutant concentrations of to the concentration the of joint molecules and exchange not The of joint molecules with The protein promoted a strand exchange with the mutant protein promoted the generation of a of reaction The in not and the in the RecA K72R reaction with and a at the the to RecA the observed for the reaction with in the not The by and RecA K72R by electron microscopy The DNA for electron microscopy with to the RecA protein to a the RecA K72R reaction the reaction molecules at strand exchange in of the molecules with the structures in and of molecules strand exchange the strand in the duplex as by the absence of duplex in the of the molecules strand in the a in and DNA molecules of the molecules in of the The molecules reaction a at the same in the of molecules at molecules a strand exchange reaction that not into the The molecules or duplex molecules of the of the and molecules of We that the RecA K72R mutant protein not a exchange exchange are not promoted by RecA as and and or RecA K72R duplex DNA substrate and of the duplex by and of The duplex the strand in the duplex by the with a are DNA by The for are and to the duplex the duplex the duplex product by a strand exchange reaction and duplex DNA with duplex and a reaction at into the reaction with and by electron The a and are in the for and reaction in the reaction with RecA a in the reaction with The to the is a of the strand exchange the reaction is as a reaction in the strand a with a strand the the duplex the by a of that is not in the exchange the the into the duplex of the a as and a strand The a and the RecA K72R at ATP as with ATP and for are in the mutant protein facilitated the The for and for the wild type RecA and the RecA K72R mutant protein, also observed in the presence of and a the presence of with wild type RecA protein and the K72R mutant in a that are to a RecA-mediated DNA strand exchange ATP hydrolysis, with Mg2+ and that the RecA DNA strand exchange is ATP is not The RecA K72R protein, does not hydrolyze or ATP at the generation of DNA with over 7 of hybrid the reaction is and greatly affected by the concentration of the reaction is also not in the duplex and not four DNA strands. of limitations are with wild type RecA protein in the presence of and and of the with the mutant protein of with wild type protein and for of the is by the in The is to the observed limitations to the of hybrid DNA RecA-mediated DNA strand exchange ATP is not to the RecA by and and Kowalczykowski, and that the is in a DNA DNA strand exchange reaction with RecA protein or ATP is to the of a the with a duplex to form a with for of a of the duplex into the with the and DNA in as in the and a DNA the length of the DNA the the hybrid DNA of strand to hybrid DNA the RecA is to DNA is at the filament, a at in the same and the to some at of DNA that by as the at the duplex DNA and at by the We the and as The of the length of a duplex The or and the reflect the of DNA a of reaction We that formation of a with and the length of the of DNA strand exchange for RecA of the and strand exchange in strand of the duplex substrate of the duplex and the of the as the to in the reaction at the the hybrid DNA that protein the of the of strand exchange in the of a DNA of the DNA in is in is at of a duplex DNA of the the of the and the duplex as by as the some for in the at the a for of the duplex DNA into the a of DNA is of the as by the form and with of the the of the RecA nucleoprotein the the of hybrid DNA formation in the of strand of the the of the the with the DNA or to the that DNA is into the at of the and of the at the a is at the of the at the of the is the that the in the the of the the the and the the as the the to the as is the in the The of migration that or by the of and some of the and the by and is with the observed of Mg2+ the reaction promoted by RecA Mg2+ concentrations a a the DNA and DNA structures and of Mg2+ in of that required to form Mg•dATP the formation of in the the formation of to to of joint of the hybrid DNA in the joint at the of of the by the Mg2+ to and the formation of The formation of structures with of the to the Mg2+ concentration to the observed in with Mg2+ and When the Mg2+ concentration is just sufficient to form the Mg•dATP complex, the the of and of completed strand exchange over of are The of Mg2+ concentration migration are in to the of Mg2+ DNA migration in DNA the formation of pairs of the at the of pairs the and the of is by to of by Mg2+ and also molecules by electron microscopy that by a that in the duplex DNA substrate is a observed at and a by of the DNA to the of the strand protein that to by of substrate duplex as in the in that type of form by at the at and of the is duplex DNA to in to is at the of the duplex and to the the the and to the to ATP is the hybrid duplex DNA is The to in to a for ATP to a of DNA to the ATP also the of exchange reaction not at ATP is in the of in DNA and that and to DNA in the absence of ATP hydrolysis, the DNA duplex DNA is at to of of RecA protein and ATP observed of Mg2+ that the with the K72R mutant are not by hybrid DNA are at Mg2+ mutant protein, in a filament, the for ATP in the exchange reaction is also in for the hybrid DNA The of RecA present ATP is not in a a exchange and a of RecA to a but at not of that the RecA DNA exchange reaction a ATP that the of RecA to hybrid DNA The that RecA-mediated ATP is to a of DNA molecules to strand exchange a to the of by a RecA that DNA RecA K72R mutant protein is in the of DNA strand exchange in the of a DNA in some to migration as or in the wild type is by a K72R a are as in as to and as to the as a mutant that the of RecA is in The in RecA is by or is RecA K72R mutant protein the to the of in in the presence of The that the K72R in by the of with the K72R mutant in the presence of ATP RecA protein of is a with a of The protein is in and is to the of DNA of the to DNA and the of at and and and RecA protein promotes a of DNA strand exchange that in and DNA The or four DNA RecA a nucleoprotein the or DNA DNA is with a duplex strand the a of hybrid is to the a strand exchange to to the DNA the of the to the RecA The reaction also a of structural barriers in the DNA protein is a with a of to wild type RecA DNA protein of ATP is the nucleoprotein When a duplex DNA is to the the to and at that the DNA strand exchange reaction as as ATP is and and The reaction is with of hybrid DNA The of ATP is by the that RecA protein DNA strand exchange some in the presence of ATP that is not by RecA and by the RecA mutant K72R and Kowalczykowski, and with wild type RecA protein in the presence of and The in the K72R mutant in a and the mutant protein binds but does not hydrolyze ATP. The mutant also a DNA strand with and Kowalczykowski, that the RecA to at DNA and DNA strand exchange ATP hydrolysis, and to for ATP in DNA strand The RecA to the hybrid DNA of a DNA strand exchange reaction does RecA protein hydrolyze to the of RecA-mediated ATP is to the limitations of that of that the strand exchange is in and bidirectional and The also not structural barriers in the DNA and and not four DNA ATP to to the DNA strand exchange required in for that barriers and for exchange some but the with of RecA-mediated DNA strand exchange in the absence of ATP ATP and strand exchange to with exchange is a formation of a of hybrid DNA product at the reaction or the is of exchange is that the reaction does not to The or of strand exchange that in some of strand exchange The to with ATP to RecA and the and Kowalczykowski, and the in a DNA that as a strand exchange and in ATP is of a duplex DNA to the of ATP by to The observed is to the length of in the that the length of is a or with over of or and strand exchange RecA the DNA or to the same to a of strand exchange in some length of hybrid DNA is by a in the hybrid DNA is The is as in hybrid DNA formation ATP is and and in the ATP is not is to the to strand exchange is the of the the of the length of the the of the and reaction and present a that the is in The also of the reaction and to with or of ATP. The a of with RecA K72R reaction and the RecA K72R mutant of the DNA strand exchange reaction promoted by the RecA K72R mutant protein but the to of the the of the RecA