Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, CSIC, Darwin 3, Campus Universidad Autónoma de Madrid, 28049 Madrid, Spain.
J Mol Biol. 2011 Jul 1;410(1):39-49. doi: 10.1016/j.jmb.2011.05.008. Epub 2011 May 12.
The Bacillus subtilis RecU protein has two activities: to recognize, distort, and cleave four-stranded recombination intermediates and to modulate RecA activities. The RecU structure shows a mushroom-like appearance, with a cap and a stalk region. The RuvB interaction and the catalytic residues are located in the cap region of dimeric RecU. We report here that the stalk region is essential not only for RecA modulation but also for Holliday junction (HJ) recognition. Two recU mutants, which map in the stalk region, were isolated and characterized. In vivo, a RecU variant with a Phe81-to-Ala substitution (F81A) was as sensitive to DNA-damaging agents as a null recU strain, and a similar substitution at tyrosine 80 (Y80A) showed an intermediate phenotype. RecUY80A and RecUF81A poorly recognize and distort HJs. RecUY80A cleaves HJs with low efficiency, and RuvB modulates cleavage. At high concentrations, RecUF81A binds to HJs but fails to cleave them. Unlike wild-type RecU, RecUY80A and RecUF81A do not inhibit RecA dATPase and strand-exchange activities. The RecU stalk region is involved in RecA interaction, but once an HJ is bound, RecU fails to modulate RecA activities. Our biochemical study provides a mechanistic basis for the connections between these two mutually exclusive stages (i.e., RecA modulation and HJ resolution) of the recombination reaction.
枯草芽孢杆菌 RecU 蛋白具有两种活性：识别、扭曲和切割四链重组中间体，以及调节 RecA 活性。RecU 结构呈蘑菇状，有帽和柄区。RuvB 相互作用和催化残基位于二聚体 RecU 的帽区。我们在此报告，柄区不仅对 RecA 调节至关重要，而且对 Holliday 连接（HJ）识别也至关重要。两个定位在柄区的 recU 突变体被分离并进行了表征。在体内，一个 F81A 取代的 RecU 变体（F81A）与无功能 recU 菌株一样对 DNA 损伤剂敏感，而 Y80A 类似取代则表现出中间表型。RecUY80A 和 RecUF81A 对 HJ 的识别和扭曲能力较差。RecUY80A 以低效率切割 HJ，并且 RuvB 调节切割。在高浓度下，RecUF81A 结合 HJ 但无法切割它们。与野生型 RecU 不同，RecUY80A 和 RecUF81A 不会抑制 RecA dATPase 和链交换活性。RecU 柄区参与 RecA 相互作用，但一旦结合 HJ，RecU 就无法调节 RecA 活性。我们的生化研究为重组反应这两个相互排斥阶段（即 RecA 调节和 HJ 解析）之间的联系提供了机制基础。
