Differential specificities and simultaneous occupancy of human MutSα nucleotide binding sites
Differential specificities and simultaneous occupancy of human MutSα nucleotide binding sites
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DOI：
10.1074/jbc.m312108200
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发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Modrich, P
中科院分区：
文献类型：
--
作者：
Martik, D;Baitinger, C;Modrich, P
我们已经检查了人类MutSalpha的允许核苷酸占用状态。MSH2.MSH6异二聚体结合1摩尔ADP和1摩尔腺苷5’-O-（硫代三磷酸）（ATP γ S），每个核苷酸的Kd约为1 μ M。使用ADP和5 '-腺苷酰-β，γ-亚氨基二磷酸（AMPPNP）的BODIPY TR和BODIPY FL荧光衍生物的各向异性测量也表明每个MutSalpha异二聚体1摩尔ADP和1摩尔三磷酸类似物的相互作用化学计量。二磷酸和三磷酸位点可以同时被占据，如通过用差异放射性标记的ADP和ATP γ S顺序填充两种结合位点类别以及通过BODIPY TR和BODIPY FL标记的ADP和AMPPNP之间的荧光共振能量转移所判断的。MutSalpha的ATP水解伴随着ADP形成的预稳态爆发，并且在第一次周转期间MutSalpha结合的核苷酸的分析已经证明了ADP和ATP两者的存在。同时存在的ADP和nonhydrolyzable ATP类似物调节MutSalpha。异源双链相互作用的方式是不同的ADP或nonhydrolyzable三磷酸单独存在下观察到的，这是不太可能的，这种效果是由于MutSalpha和ADP或三磷酸类似物之间的二元复合物的混合人口的存在。这些结果表明MutSalpha具有两个对ADP和ATP具有不同特异性的核苷酸结合位点，并表明ADP·MutSalpha·ATP三元复合物在错配修复中具有重要作用。
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 muM. 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.