Hegde S P, Rajagopalan M, Madiraju M V
Department of Microbiology, University of Texas Health Center at Tyler 75710, USA.
J Bacteriol. 1996 Jan;178(1):184-90. doi: 10.1128/jb.178.1.184-190.1996.
Escherichia coli RecF protein binds, but does not hydrolyze, ATP. To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped [g]DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog. Protein-DNA complexes were analyzed by electrophoresis on agarose gels and visualized by autoradiography. The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substrates and from those formed in the absence of gamma-S-ATP. Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP, and the order of preference of binding was gDNA > dsDNA > ssDNA. Since gDNA has both ds- and ssDNA components, we suggest that the role for ATP in RecF protein-DNA interactions in vivo is to confer specificity of binding to dsDNA-ssDNA junctions, which is necessary for catalyzing DNA repair and recombination.
大肠杆菌RecF蛋白能够结合ATP，但不会水解ATP。为了确定ATP结合到RecF上在RecF蛋白介导的DNA结合中所起的作用，我们测定了RecF蛋白与单链（ss）DNA、双链（ds）DNA以及含有单链DNA区域的双链DNA（缺口[g]DNA）之间的相互作用，这些DNA单独存在或组合存在，且分别处于有和没有腺苷（γ-硫代）三磷酸（γ-S-ATP，一种不可水解的ATP类似物）的情况下。通过琼脂糖凝胶电泳分析蛋白质-DNA复合物，并通过放射自显影进行可视化。在γ-S-ATP存在下形成的蛋白质-DNA复合物类型因每种DNA底物而异，且与在没有γ-S-ATP时形成的复合物不同。用各种DNA底物组合进行的竞争实验表明，在γ-S-ATP存在下，RecF蛋白优先结合缺口DNA，结合偏好顺序为缺口DNA＞双链DNA＞单链DNA。由于缺口DNA同时具有双链和单链DNA成分，我们认为ATP在体内RecF蛋白与DNA相互作用中的作用是赋予与双链DNA-单链DNA连接处结合的特异性，这对于催化DNA修复和重组是必需的。
