Kowalczykowski S C
Biochemistry. 1986 Oct 7;25(20):5872-81. doi: 10.1021/bi00368a006.
The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied. The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity. Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine. Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP. In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased. These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa. In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity. The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed.
已对ATP光亲和类似物8-叠氮腺苷5'-三磷酸（叠氮-ATP）与recA蛋白的结合及交联进行了研究，并且通过交联抑制研究，也对其他核苷酸辅因子与recA蛋白的结合进行了研究。就recA蛋白结合和酶功能而言，叠氮-ATP分子被证明是一种良好的ATP类似物，依据以下三条标准：第一，交联遵循简单的双曲线结合曲线，解离常数（Kd）为4微摩尔，交联效率根据条件在10%至70%之间；第二，ATP、dATP和腺苷5'-O-(3-硫代三磷酸)（ATP-γ-S）特异性抑制叠氮-ATP与recA蛋白的交联；第三，叠氮-ATP是recA蛋白ATP酶活性的底物。使用多种核苷酸辅因子作为竞争者对交联抑制研究进行的定量分析表明，含腺嘌呤的核苷酸对recA蛋白的结合亲和力按以下顺序降低：ATP-γ-S＞dATP＞ATP＞腺苷β,γ-亚氨基二磷酸（AMP-PNP）＞＞腺苷β,γ-亚甲基二磷酸（AMP-PCP）≈腺嘌呤。类似的竞争研究还表明，几乎所有其他三磷酸核苷酸也与recA蛋白结合，亲和力按以下顺序降低：UTP＞GTP≈dCTP＞dGTP＞CTP。此外，在单链DNA存在下进行的研究表明，ATP、dATP、ATP-γ-S和AMP-PNP对recA蛋白的亲和力显著增加。根据核苷酸辅因子对recA蛋白与单链DNA结合亲和力调节的相互作用以及反之亦然的情况，对这些结果进行了讨论。此外，已证明核苷酸和DNA结合是ATP酶活性的必要条件，但不是充分条件。讨论了该结果对于ATP酶活性可能需要15个或更多recA蛋白分子连续结合到DNA上的临界大小簇的意义。
