The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing
The mutant RecA proteins, RecAR243Q and RecAK245N, exhibit defective DNA binding in homologous pairing
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DOI：
10.1006/abbi.1999.1166
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发表时间：
1999-05-01
影响因子：
3.9
通讯作者：
Shibata, T
中科院分区：
文献类型：
--
作者：
Kurumizaka, H;Ikawa, S;Shibata, T
在同源配对中，RecA蛋白依次与单链DNA（ssDNA）和双链DNA（dsDNA）结合，使螺旋状核蛋白丝内的两个DNA分子对齐。为了确定从丝的外侧延伸到内侧的DNA结合区域，我们构建了两种突变的RecA蛋白，RecAR243Q和RecAK245N，分别将精氨酸243替换为谷氨酰胺，赖氨酸245替换为天冬酰胺。这些氨基酸在RecA蛋白的晶体结构中暴露于溶剂中，并且位于中心结构域，该结构域被认为是同源配对活性的催化中心。精氨酸243替换为谷氨酰胺（RecAR243Q）以及赖氨酸245替换为天冬酰胺（RecAK245N）会损害体内紫外线损伤DNA的修复，并导致体外ssDNA和dsDNA的同源配对缺陷。尽管RecAR243Q仅有轻微缺陷，RecAK245N在ssDNA结合形成突触前丝方面完全正常，但两种突变的RecA蛋白在包括ssDNA、dsDNA和RecA蛋白的三组分复合物的形成方面均存在缺陷。RecAR243Q和RecAK245N在由互补单链形成dsDNA的能力方面也存在缺陷。这些结果表明，包含精氨酸243和赖氨酸245的区域可能参与二次DNA与突触前丝结合的路径。（C）1999年学术出版社
In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gin and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gin (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNA in vivo and cause defective homologous pairing of ssDNA and dsDNA in vitro. Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein, The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament. (C) 1999 Academic Press.