Stole E, Bryant F R
Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205, USA.
J Biol Chem. 1995 Sep 1;270(35):20322-8. doi: 10.1074/jbc.270.35.20322.
We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.
我们最近构建了一种突变型RecA蛋白，其中His-163被色氨酸残基取代；[H163W]RecA蛋白在功能上与野生型蛋白相同，并且Trp-163侧链作为[H163W]RecA-单链DNA（ssDNA）复合物构象转变的报告基团。我们现在研究了在一系列替代核苷三磷酸辅因子存在下[H163W]RecA-ssDNA复合物的荧光特性。在标准条件（pH 7.5）下，ATP（S0.5 = 70 microM）和嘌呤核糖三磷酸（PTP）（S0.5 = 110 microM）使Trp-163荧光降低44%，并作为DNA链交换反应的辅因子具有活性。相比之下，ITP（S0.5 = 400 microM）仅使Trp-163荧光降低20%（与核苷二磷酸ADP、PDP和IDP观察到的水平相同），并且作为链交换辅因子无活性。如果将S0.5（PTP）增加到130 microM（通过提高反应溶液的pH值），PTP介导的Trp-163荧光猝灭降至20%，并且PTP作为链交换辅因子变得无活性。这些结果为核苷三磷酸的S0.5值与RecA-ssDNA复合物的构象状态之间的联系提供了直接证据，稳定链交换活性构象需要100 - 120 microM或更低的S0.5。
