Differential and Simultaneous Adenosine Di- and Triphosphate Binding by MutS

Keith P Bjornson,Paul Modrich

doi:10.1074/jbc.m301101200

Keith P Bjornson, Paul Modrich

Open Access

https://doi.org/10.1074/jbc.m301101200

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Abstract

The roles of ATP binding and hydrolysis in the function of MutS in mismatch repair are poorly understood. As one means of addressing this question, we have determined the affinities and number of adenosine di- and triphosphate binding sites within MutS. Nitrocellulose filter binding assay and equilibrium fluorescence anisotropy measurements have demonstrated that MutS has one high affinity binding site for ADP and one high affinity site for nonhydrolyzable ATP analogues per dimer equivalent. Low concentrations of 5'-adenylylimidodiphosphate (AMPPNP) promote ADP binding and a large excess of AMPPNP is required to displace ADP from the protein. Fluorescence energy transfer and filter binding assays indicate that ADP and nonhydrolyzable ATP analogues can bind simultaneously to adjacent subunits within the MutS oligomer with affinities in the low micromolar range. These findings suggest that the protein exists primarily as the ATP.MutS.ADP ternary complex in solution and that this may be the form of the protein that is involved in DNA encounters in vivo.

Highlights

Mismatch repair contributes genetic stability by correcting DNA biosynthetic errors and preventing recombination between quasi-homologous DNA sequences [1,2,3,4,5]
Structural studies have shown bacterial MutS [13,14,15] and the DNA repair protein RAD50 [16] to be members of the adenine nucleotide binding cassette (ABC)1 family of proteins, which is largely composed of proteins that couple the energy of ATP hydrolysis to transport of molecules across biological membranes [17, 18]
MutS Binds ADP and AMPPNP with Similar Affinities— Nucleotide binding by MutS was examined by filter assay using radiolabeled ADP and nonhydrolyzable AMPPNP and by fluorescent anisotropy determination using nucleotides chemically modified with the BODIPY (FL or TR) fluorophore on the 2Ј or 3Ј positions of the ribose moiety

Summary

MutS Nucleotide Binding Site Occupancy

The finding that ATP promotes release of MutS homologs from a mismatch (22, 25–28, 34 –36) and the observation that mismatch specificity of bacterial MutS is abolished in the presence of a non-hydrolyzable ATP analogue [29] are seemingly incompatible with these ideas To some extent, these disparate models reflect the lack of information concerning the effects of ATP binding and hydrolysis and, potential states of nucleotide occupancy on MutS-DNA interaction. Recent work indicates that MutS dimers undergo a concentration-dependent assembly reaction that is promoted by binding of nucleotide or heteroduplex DNA, probably to a tetramer end state [14, 24].2, 3 This dramatically increases the complexity of the system. We show that MutS binds ADP and non-hydrolyzable ATP analogues with similar affinities, that the protein has one high affinity ADP site and one high affinity triphosphate site per dimer equivalent, and that di- and triphosphate binding sites can be simultaneously occupied

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION