Alonso J C, Stiege A C, Dobrinski B, Lurz R
Max-Planck-Institut für molekulare Genetik, Berlin, Germany.
J Biol Chem. 1993 Jan 15;268(2):1424-9.
Genetic evidence suggests that the Bacillus subtilis recR gene product is involved in DNA repair and recombination. To assign a biochemical function to the recR gene product, the RecR protein was labeled and purified by monitoring the radioactive label. NH2-terminal protein sequence analysis of RecR was consistent with the deduced amino acid sequence of the recR gene. The RecR protein (molecular mass of 25 kDa, isoelectric point 5.4) bound single- and double-stranded DNA in a filter binding assay. RecR-DNA complex formation is enhanced by the presence of a damage in the DNA substrate. The RecR-DNA complex formation proceeds in the absence of divalent cations and nucleotide cofactors, but is markedly stimulated by ATP and divalent cations. In our experimental conditions the apparent equilibrium constants of the optimized RecR-DNA complexes are 3 x 10(-7) M and 9 x 10(-7) M for damaged and undamaged DNA, respectively. The binding reaction is cooperative. Electron microscopy studies show that the presence of divalent cations increases the rate of RecR protein self-assembly. Addition of ATP or dATP promotes the organization of discrete series of quaternary structures on DNA, but ATP gamma S inhibits the DNA binding activity. A possible mechanism for the RecR function in DNA repair is discussed.
遗传证据表明，枯草芽孢杆菌recR基因产物参与DNA修复和重组。为了赋予recR基因产物生化功能，通过监测放射性标记对RecR蛋白进行标记和纯化。RecR的氨基末端蛋白质序列分析与recR基因推导的氨基酸序列一致。在滤膜结合试验中，RecR蛋白（分子量25 kDa，等电点5.4）能结合单链和双链DNA。DNA底物中存在损伤会增强RecR-DNA复合物的形成。RecR-DNA复合物的形成在没有二价阳离子和核苷酸辅因子的情况下也能进行，但ATP和二价阳离子会显著刺激其形成。在我们的实验条件下，优化后的RecR-DNA复合物对于损伤DNA和未损伤DNA的表观平衡常数分别为3×10⁻⁷ M和9×10⁻⁷ M。结合反应具有协同性。电子显微镜研究表明，二价阳离子的存在会增加RecR蛋白的自组装速率。添加ATP或dATP会促进DNA上离散系列四级结构的形成，但ATPγS会抑制DNA结合活性。文中还讨论了RecR在DNA修复中发挥功能的可能机制。
