Mutations within the hMLH1 and hPMS2 Subunits of the Human MutLα Mismatch Repair Factor Affect Its ATPase Activity, but Not Its Ability to Interact with hMutSα

Abstract

The MutL family of mismatch repair proteins belongs to the GHKL class of ATPases, which contains also type II topoisomerases, HSP90, and histidine kinases. The nucleotide binding domains of these polypeptides are highly conserved, but this similarity has failed to help us understand the biological role of the ATPase activity of the MutL proteins in mismatch repair. hMutLα is a heterodimer of the human MutL homologues hMLH1 and hPMS2, and we decided to exploit its asymmetry to study this function. We now show that although the two subunits contribute differently to the ATPase activity of the heterodimer, hMutLα variants in which one subunit to but mismatch repair in variants in which subunit to the nucleotide of the of subunits repair the of these to one the binding of the nucleotide in hMutLα the of the mismatch to a we that the ATPase activity of hMutLα is this The MutL family of mismatch repair proteins belongs to the GHKL class of ATPases, which contains also type II topoisomerases, HSP90, and histidine kinases. The nucleotide binding domains of these polypeptides are highly conserved, but this similarity has failed to help us understand the biological role of the ATPase activity of the MutL proteins in mismatch repair. hMutLα is a heterodimer of the human MutL homologues hMLH1 and hPMS2, and we decided to exploit its asymmetry to study this function. We now show that although the two subunits contribute differently to the ATPase activity of the heterodimer, hMutLα variants in which one subunit to but mismatch repair in variants in which subunit to the nucleotide of the of subunits repair the of these to one the binding of the nucleotide in hMutLα the of the mismatch to a we that the ATPase activity of hMutLα is this mismatch repair mismatch repair is a highly that the repair of that but also that the repair to the and the that the to the and are the which the repair the of the the MutL the and II one of and and The MutL a role in this the activity of which the is the of in and the of to the MutL also to of II the of the The is and the is the and the that the of the of the of the is is the MutL the role of which is is is that its of are binding of that MutL and a in and in of and that MutL in the of the binding of MutL to the of the of MutL to that are also in the type II topoisomerases, the proteins and histidine of of that these proteins a and role the in binding of a this of GHKL ATPases, is to a the of these is a of biological of the of MutL that the two domains The that the nucleotide binding the a is to a that a in the in to nucleotide binding the and the MutL of and of the MutL that its binding of binding of of in in The of that this and to nucleotide binding to the of the that the of MutL this the of MutL II of a to a of in to a and of has to a role in the polypeptides the is in the of the in its that MutL to a role of binding the that the binding of MutL that the to MutL to the ATPase activity of MutL of the binding and the of the ATPase activity of to highly conserved, the of and MutL homologues in to human the homologues and two and which are in mismatch the homologues hPMS2, and to in the human hMLH1 and a role in The polypeptides these to a heterodimer, which is in in the to of the and the in the the in the of which of these are in the highly that the ATPase of the hMLH1 in the in this to that the is to the that the of is to a of the MutL that in are We to the the of the binding domains of the two subunits of hMutLα in the We show that to in the of human in the hMLH1 the we of this the ATPase of hMLH1 and and the of the variants in and of to the of the the of the type proteins in to MutL homologues that in the binding the and in a the The the and in a the The the the and in a the The the and in a the The the the and in a the The the and in a the The the the of the and and The the and of the and in a the The the and in a the The the the and in a the The the and in a the The the of the and in a the The of the of the and the of the and of the hMutLα a of two the hMLH1 and variants a of of the of the in of and the in a of a and a of in a and in of the hMutLα of and in a to a and proteins a the hMutLα variants to of of a of The of proteins of and of the highly variants to and a to the the proteins a to the and proteins a The the proteins and and in of in of and The in and in and a of of the proteins in the of The and in a of and the to The the of of and The and the and binding a in the hMLH1 of type hMutLα of and The proteins of and the and The in the of is the of the is the of and The the of which is to in the of the hMLH1 that the the ATPase in a and of the of the hMutLα heterodimer, a and to the and of which to The the of the the ATPase activity in the of of and in the in a of the to in a and a of the and the and in type and to the The the the of the and a the of of of the hMutLα The repair and and of and of the and in a of a of of and of repair the of the a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the the of MutL homologues in the human the heterodimer hMutLα the role in is the that human in of this that subunits of hMutLα are the that the in and that the two are to that are The of to the in the that the the two the of the of hMLH1 and to the of the the that the to its and in the of the hMutLα the in the binding of hPMS2, which to the that binding in the hMLH1 subunit the of the heterodimer to hMutLα variants in that to the binding of hMLH1 and of the to binding to the of the that the to is the of hMLH1 but and that the two polypeptides the the of the of to hMLH1 We that the binding of hMLH1 to and that is the of the the of this to the of binding to hMLH1 to that the to the type we that the of this but this the of binding in the to the but to its biological activity also the and that the hMLH1 subunit of hMutLα the nucleotide of binding in the subunit the ATPase activity of the heterodimer to a the in the hMLH1 that is hMLH1 and that the of the nucleotide in the binding of are of and that the of has ATPase the in ATPase of the two hMutLα subunits the biological activity of the hMutLα variants we the in in the and that to to the of The the subunit that in nucleotide show that binding in subunits is the and hMutLα in which the in one the but in that the ATPase of the two the to and that hMutLα to in to in but that the activity we a we the binding of the and subunits of the subunit the ATPase activity of the heterodimer, the of in and mismatch repair in this study in the of the ATPase domains of of The to the to in the binding of the proteins in and and to to nucleotide nucleotide the in which a of the a of a subunit a in the the and this asymmetry that the to the a a that binding is and II variants that the in the of the the of the to that of the in The and and that of the the of the to a which the of nucleotide asymmetry the to of the two subunits the repair a a the two subunits to and the two binding domains to of We this the repair of the that the the the of the The that these to a that the to the human and that the two binding domains of hMutLα in a is and the of which that binding subunits is in the nucleotide binding domains to that the of the proteins the of the to which the the in this study us to a hMutLα in which binding a the of its two The binding of the hMLH1 subunit that of the subunit the and the and that the proteins the nucleotide binding of the binding of the which in of the and in of hMutLα its of The heterodimer the of the which to and of the of the of of variants to one of subunits to the the The in of the also we that one subunit to the of the and to the repair but biological role of the MutL homologues has that of the MutL that a that the to the MutL to the II and to the of the the of of is the the activity of which to MutL in a that binding but is in the of and a but in this the ATPase activity of MutL is the of the MutL which but to to the of the of in that of the role of the MutL homologues in in the that and in The human are of and study that the and of the hMutLα binding the hMutLα the of the in the of hMutLα that the of and is binding this heterodimer to that of nucleotide the show that the hMutLα variants that to We that the of activity of these proteins in in to to of the that are mismatch mismatch repair is a highly that the repair of that but also that the repair to the and the that the to the and are the which the repair the of the the MutL the and II one of and and The MutL a role in this the activity of which the is the of in and the of to the MutL also to of II the of the The is and the is the and the that the of the of the of the is is the MutL the role of which is is is that its of are binding of that MutL and a in and in of and that MutL in the of the binding of MutL to the of the of MutL to that are also in the type II topoisomerases, the proteins and histidine of of that these proteins a and role the in binding of a this of GHKL ATPases, is to a the of these is a of biological function. The of the of MutL that the two domains The that the nucleotide binding the a is to a that a in the in to nucleotide binding the and the MutL of and of the MutL that its binding of binding of of in in The of that this and to nucleotide binding to the of the that the of MutL this the of MutL II of a to a of in to a and of has to a role in the polypeptides the is in the of the in its that MutL to a role of binding the that the binding of MutL that the to MutL to the ATPase activity of MutL of the binding and the of the ATPase activity The of to highly conserved, the of and MutL homologues in to human the homologues and two and which are in mismatch the homologues hPMS2, and to in the human hMLH1 and a role in The polypeptides these to a heterodimer, which is in in the to of the and the in the the in the of which of these are in the highly that the ATPase of the hMLH1 in the in this to that the is to the that the of is to a of the MutL that in are We to the the of the binding domains of the two subunits of hMutLα in the We show that to in the of human in the hMLH1 the we of this the ATPase of hMLH1 and and the of the variants in and of to the of the the of the type proteins in to MutL homologues that in the binding the and in a the The the and in a the The the the and in a the The the and in a the The the the and in a the The the and in a the The the the of the and and The the and of the and in a the The the and in a the The the the and in a the The the and in a the The the of the and in a the The of the of the and the of the and of the hMutLα a of two the hMLH1 and variants a of of the of the in of and the in a of a and a of in a and in of the hMutLα of and in a to a and proteins a the hMutLα variants to of of a of The of proteins of and of the highly variants to and a to the the proteins a to the and proteins a The the proteins and and in of in of and The in and in and a of of the proteins in the of The and in a of and the to The the of of and The and the ATPase in a and of the of the hMutLα heterodimer, a and to the and of which to The the of the the ATPase activity in the of of and in the in a of the to in a and a of the and the and in type and to the The the the of the and a the of of of the hMutLα The repair and and of and of the and in a of a of of and of repair the of the a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the and of to the of the the of the type proteins in to MutL homologues that in the binding the and in a the The the and in a the The the the and in a the The the and in a the The the the and in a the The the and in a the The the the of the and and The the and of the and in a the The the and in a the The the the and in a the The the and in a the The the of the and in a the The of the of the and the of the The to the of the the of the type proteins in to MutL homologues that in the binding the and in a the The the and in a the The the the and in a the The the and in a the The the the and in a the The the and in a the The the the of the and and The the and of the and in a the The the and in a the The the the and in a the The the and in a the The the of the and in a the The of the of the and the of the the and in a the The the and in a the The the and in a the The the the and in a the The the the and in a the The the and in a the The the and in a the The the the and in a the The the the and in a the The the and in a the The the and in a the The the the and in a the The the the of the and and The the and of the of the and and The the and of the and in a the The the and in a the The the and in a the The the the and in a the The the the and in a the The the and in a the The the and in a the The the of the and in a the The the of the and in a the The the and in a the The of the of the and the of the of the of the and the of the and and of the hMutLα a of two the hMLH1 and variants a of of the of the in of and the in a of a and a of in a and in a of two the hMLH1 and variants a of of the of the in of and the in a of a and a of in a and in of the hMutLα of and in a to a and proteins a the hMutLα variants to of of a of The of proteins of and of the highly variants to and a to the the proteins a to the and proteins a The the proteins and and in of and in a to a and proteins a the hMutLα variants to of of a of The of proteins of and of the highly variants to and a to the the proteins a to the and proteins a The the proteins and and in The of in of and The in and in and of in of and The in and in and a of of the proteins in the of The and in a of and the to The the of of and The and the a of of the proteins in the of The and in a of and the to The the of of and The and the ATPase ATPase in a and of the of the hMutLα heterodimer, a and to the and of which to The the of the the ATPase activity in the of of The ATPase in a and of the of the hMutLα heterodimer, a and to the and of which to The the of the the ATPase activity in the of of and in the in a of the to in a and a of the and the and in type and to the The the the of the and a the of of of the hMutLα The repair and and of and of the and in a of a of of and of repair the of the a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the The and in the in a of the to in a and a of the and the and in type and to the The the the of the and a the of of of the hMutLα The repair and and of and of the and in a of a of of and of repair the of the a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the The a a a The and hMutLα variants in of binding to the of the to in which hMLH1 the of the of a in and a The and the the that its the the of MutL homologues in the human the heterodimer hMutLα the role in is the that human in of this that subunits of hMutLα are the that the in and that the two are to that are The of to the in the that the the two the of the of hMLH1 and to the of the the that the to its and in the of the hMutLα the in the binding of hPMS2, which to the that binding in the hMLH1 subunit the of the heterodimer to hMutLα variants in that to the binding of hMLH1 and of the to binding to the of the that the to is the of hMLH1 but and that the two polypeptides the the of the of to hMLH1 We that the binding of hMLH1 to and that is the of the the of this to the of binding to hMLH1 to that the to the type we that the of this but this the of binding in the to the but to its biological activity also the and that the hMLH1 subunit of hMutLα the nucleotide of binding in the subunit the ATPase activity of the heterodimer to a the in the hMLH1 that is hMLH1 and that the of the nucleotide in the binding of are of and that the of has ATPase the in ATPase of the two hMutLα subunits the biological activity of the hMutLα variants we the in in the and that to to the of The the subunit that in nucleotide show that binding in subunits is the and hMutLα in which the in one the but in that the ATPase of the two the to and that hMutLα to in to in but that the activity we a we the binding of the and subunits of the subunit the ATPase activity of the heterodimer, the of in and mismatch repair in this study in the of the ATPase domains of of The to the to in the binding of the proteins in and and to to nucleotide nucleotide the in which a of the a of a subunit a in the the and this asymmetry that the to the a a that binding is and II variants that the in the of the the of the to that of the in The and and that of the the of the to a which the of nucleotide asymmetry the to of the two subunits the repair a a the two subunits to and the two binding domains to of We this the repair of the that the the the of the The that these to a that the to the human and that the two binding domains of hMutLα in a is and the of which that binding subunits is in the nucleotide binding domains to that the of the proteins the of the to which the the in this study us to a hMutLα in which binding a the of its two The binding of the hMLH1 subunit that of the subunit the and the and that the proteins the nucleotide binding of the binding of the which in of the and in of hMutLα its of The heterodimer the of the which to and of the of the of of variants to one of subunits to the the The in of the also we that one subunit to the of the and to the repair but biological role of the MutL homologues has that of the MutL that a that the to the MutL to the II and to the of the the of of is the the activity of which to MutL in a that binding but is in the of and a but in this the ATPase activity of MutL is the of the MutL which but to to the of the of in that of the role of the MutL homologues in in the that and in The human are of and study that the and of the hMutLα binding the hMutLα the of the in the of hMutLα that the of and is binding this heterodimer to that of nucleotide the show that the hMutLα variants that to We that the of activity of these proteins in in to to of the that are mismatch the of MutL homologues in the human the heterodimer hMutLα the role in is the that human in of this that subunits of hMutLα are the that the in and that the two are to that are The of to the in the that the the two the of the of hMLH1 and to the of the the that the to its and in the of the hMutLα the in the binding of hPMS2, which to the that binding in the hMLH1 subunit the of the heterodimer to hMutLα variants in that to the binding of hMLH1 and of the to binding to the of the that the to is the of hMLH1 but and that the two polypeptides the the of the of to hMLH1 We that the binding of hMLH1 to and that is the of the the of this to the of binding to hMLH1 to that the to the type we that the of this but this the of binding in the to the but to its biological activity also the and that the hMLH1 subunit of hMutLα the nucleotide of binding in the subunit the ATPase activity of the heterodimer to a the in the hMLH1 that is hMLH1 and that the of the nucleotide in the binding of are of and that the of has ATPase the in ATPase of the two hMutLα subunits the biological activity of the hMutLα variants we the in in the and that to to the of The the subunit that in nucleotide show that binding in subunits is the and hMutLα in which the in one the but in that the ATPase of the two the to and that hMutLα to in to in but that the activity we a we the binding of the and subunits of the subunit the ATPase activity of the heterodimer, the of in and mismatch repair in this study in the of the ATPase domains of of The to the to in the binding of the proteins in and and to to nucleotide nucleotide the in which a of the a of a subunit a in the the and this asymmetry that the to the a a that binding is and II variants that the in the of the the of the to that of the in The and and that of the the of the to a which the of nucleotide The asymmetry the to of the two subunits the repair a a the two subunits to and the two binding domains to of We this the repair of the that the the the of the The that these to a that the to the human and that the two binding domains of hMutLα in a is and the of which that binding subunits is in the nucleotide binding domains to that the of the proteins the of the to which the the in this study us to a hMutLα in which binding a the of its two The binding of the hMLH1 subunit that of the subunit the and the and that the proteins the nucleotide binding of the binding of the which in of the and in of hMutLα its of The heterodimer the of the which to and of the of the of of variants to one of subunits to the the The in of the also we that one subunit to the of the and to the repair but The biological role of the MutL homologues has that of the MutL that a that the to the MutL to the II and to the of the the of of is the the activity of which to MutL in a that binding but is in the of and a but in this the ATPase activity of MutL is the of the MutL which but to to the of the of in that of the role of the MutL homologues in in the that and in The human are of and study that the and of the hMutLα binding the hMutLα the of the in the of hMutLα that the of and is binding this heterodimer to that of nucleotide the show that the hMutLα variants that to We that the of activity of these proteins in in to to of the that are mismatch We and the and help the We also and