Differential specificities and simultaneous occupancy of human MutSalpha nucleotide binding sites.
We have examined the permissible nucleotide occupancy states of human MutSalpha. The MSH2.MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5'-O-(thiotriphosphate) (ATPgammaS), with a K(d) for each nucleotide of about 1 microm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSalpha heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPgammaS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSalpha is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSalpha-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSalpha.heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSalpha and ADP or a triphosphate analogue. These findings imply that MutSalpha has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP.MutSalpha.ATP ternary complex has an important role in mismatch repair.
Martik
Diana
D
Department of Biochemistry and Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
Baitinger
Celia
C
Modrich
Paul
P
D000225:Adenine / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D016880:Anisotropy; D020137:Base Pair Mismatch; D001665:Binding Sites; D019921:Biotinylation; D001896:Boron Compounds / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008856:Microscopy, Fluorescence; D051718:MutS Homolog 2 Protein; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013997:Time Factors
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C102997:Msh6 protein, mouse; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein; D000225:Adenine