Differential Specificities and Simultaneous Occupancy of Human MutSα Nucleotide Binding Sites

Diana Martik,Celia Baitinger,Paul Modrich

doi:10.1074/jbc.m312108200

Diana Martik, Celia Baitinger + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m312108200

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Abstract

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.

Highlights

Mismatch repair corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination, and participates in the cellular response to certain types of DNA damage [1,2,3,4,5,6]
Using filter binding assay and fluorescent methods, we have examined the interaction of MutS␣ with ADP, adenosine 5؅-O-(thiotriphosphate) (ATP␥S), AMPPNP, and the BODIPY fluorescent derivatives of ADP and AMPPNP
The Kd value for ATP␥S is in agreement with that obtained by Gradia et al [28]; these results show that MutS␣ binds ADP and the nonhydrolyzable ATP analogue AMPPNP with similar high affinities

Summary

EXPERIMENTAL PROCEDURES

Proteins and Oligonucleotides—Human MutS␣ was purified from SF9 cells infected with a baculovirus expressing construct by a modification of the previously described procedure [7, 36]. To evaluate the significance of ATP␥S hydrolysis, 8 ␮M MutS␣ was incubated under filter binding conditions (0 °C in binding buffer) with 8 ␮M [35S]ATP␥S, the reaction was sampled as a function of time, samples were analyzed by chromatography on PEI-cellulose plates (EM Science, Gibbstown, NJ) as described [40], and radioactivity was quantitated using an Amersham Biosciences PhosphorImager. This analysis indicated that hydrolysis under filter-binding conditions occurred at a rate of 1.6% per min. This R0 value and the measured efficiency of energy transfer were used to estimate the distance between di- and triphosphate binding centers of MutS␣ as described [43]

RESULTS

KDKTM KDKT ϩ KTDtotal ϩ KDT

None ADP

DISCUSSION