Alexseyev A A, Bakhlanova I V, Zaitsev E N, Lanzov V A
Division of Molecular and Radiation Biophysics, B. P. Konstantinov Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina/St. Petersburg, Russia.
J Bacteriol. 1996 Apr;178(7):2018-24. doi: 10.1128/jb.178.7.2018-2024.1996.
To search for functionally thermosensitive (FT) recA mutations, as well as mutations with differently affect RecA protein functions, seven new recA mutations in three different regions of the RecA protein structure proposed by Story et al. [R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992] were constructed. Additionally, the recA2283 allele responsible for the FT phenotype of the recA200 mutant was sequenced. Five single mutations (recA2277, recA2278, recA2283, recA2283E, and recA2284) and one double mutation (recA2278-5) generated, respectively, the amino acid substitutions L-277-->N, G-278-->P, L-283-->P, L-283-->E, I-284-->D, and G-278-->T plus V-275-->F in the alpha-helix H-beta-strand 9 region of the C-terminal domain of the RecA protein structure. According to recombination, repair, and SOS-inducible characteristics, these six mutations fall into four phenotypic classes: (i) an FT class, with either inhibition of all three analyzed functions at 42 degrees C (recA2283), preferable inhibition at 42 degrees C of recombination and the SOS response (recA2278), or inhibition at 42 degrees C of only recombination (recA2278-5); (ii) a moderately deficient class (recA2277); (iii) a nondeficient class (recA2283E); and (iv) a mutation with a null phenotype (recA2284). The recA2223 mutation generates an L-223-->M substitution in beta-strand 6 in a central domain of the RecA structure. This FT mutation shows preferable inhibition of the SOS response at 42 degrees C. The recA2183 mutation produces a K-183-->M substitution in alpha-helix F of the same domain. The Lys-183 position in the Escherichia coli RecA protein was found among positions which are important for interfilament interaction (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992).
为了寻找功能上对温度敏感（FT）的recA突变，以及对RecA蛋白功能有不同影响的突变，构建了Story等人[R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318 - 325, 1992]提出的RecA蛋白结构三个不同区域中的7个新的recA突变。此外，对导致recA200突变体FT表型的recA2283等位基因进行了测序。产生的5个单突变（recA2277、recA2278、recA2283、recA2283E和recA2284）和1个双突变（recA2278 - 5）分别在RecA蛋白结构C端结构域的α - 螺旋H - β - 链9区域产生了氨基酸替换L - 277→N、G - 278→P、L - 283→P、L - 283→E、I - 284→D，以及G - 278→T加V - 275→F。根据重组、修复和SOS诱导特性，这6个突变分为4个表型类别：（i）FT类别，在42℃时对所有3种分析功能均有抑制作用（recA2283），在42℃时对重组和SOS应答有优先抑制作用（recA2278），或在42℃时仅对重组有抑制作用（recA2278 - 5）；（ii）中度缺陷类别（recA2277）；（iii）无缺陷类别（recA2283E）；（iv）具有无效表型的突变（recA2284）。recA2223突变在RecA结构中心结构域的β - 链6中产生L - 223→M替换。这个FT突变在42℃时对SOS应答有优先抑制作用。recA2183突变在同一结构域的α - 螺旋F中产生K - 183→M替换。在大肠杆菌RecA蛋白中的Lys - 183位置位于对丝间相互作用很重要的位置之中（R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318 - 325, 1992）。
