Saturation mutagenesis of the E-coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair
Saturation mutagenesis of the E-coli RecA loop L2 homologous DNA pairing region reveals residues essential for recombination and recombinational repair
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DOI：
10.1006/jmbi.1998.2515
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发表时间：
1999-03-05
影响因子：
5.6
通讯作者：
Camerini-Otero, RD
中科院分区：
文献类型：
--
作者：
Hörtnagel, K;Voloshin, ON;Camerini-Otero, RD
已知大肠杆菌RecA蛋白的无序移动环L2在DNA结合和配对中起核心作用。为了研究与功能相关的局部化学环境，我们使用位点定向诱变程序对环L2区域（氨基酸位置193-212）进行了饱和诱变，并使用同源重组和重组修复的基因分析确定了380个突变体的重组能力。残基Asn193、Gln194、Arg196、Glu207、Thr209、Gly211和Gly212被鉴定为细菌细胞重组事件的严格必需。此外，我们的研究结果表明，环L2参与了RecA的ATP酶活性，残基Gln194、Arg196、Lys198和Thr209在ATP的dna依赖性水解中发挥了作用。最后，由于包含该区域的20个残基肽可以通过形成丝状β结构对同源dna进行配对，我们提出了如何利用突变分析的信息促进使用简化的氨基酸字母表来设计β结构，形成具有改进的reca样活性的L2肽。
The disordered mobile loop L2 of the Escherichia coli RecA protein is known to play a central role in DNA binding and pairing. To investigate the local chemical environment in relation to function we performed saturation mutagenesis of the loop L2 region (amino acid positions 193-212) using a site-directed mutagenesis procedure, and determined the recombinational proficiency of the 380 mutants using genetic assays for homologous recombination and recombinational repair. Residues Asn193, Gln194, Arg196, Glu207, Thr209, Gly211, and Gly212 were identified as stringently required for recombinational events in bacterial cells. In addition, our findings suggest the involvement of loop L2 in the ATPase activity of RecA, and a role for residues Gln194, Arg196, Lys198 and Thr209 in the DNA-dependent hydrolysis of ATP. Finally, since 20 residue peptides that comprise this region can pair homologous DNAs by forming filamentous beta-structures, we propose how the information from the mutant analysis might facilitate the use of a simplified amino acid alphabet to design beta-structure forming L2 peptides with improved RecA-like activities.