Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2-Msh6 mismatch repair protein.
Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1-but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.
Antony
Edwin
E
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, CT 06459, USA.
Khubchandani
Sapna
S
Chen
Siying
S
Hingorani
Manju M
MM
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry; D005800:Genes, Fungal; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D051718:MutS Homolog 2 Protein / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013997:Time Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C098790:MSH6 protein, S cerevisiae; D009711:Nucleotides; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C489751:MSH2 protein, S cerevisiae; D051719:MutS DNA Mismatch-Binding Protein; D051718:MutS Homolog 2 Protein