Namsaraev E A, Baitin D, Bakhlanova I V, Alexseyev A A, Ogawa H, Lanzov V A
Division of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina/St Petersburg.
Mol Microbiol. 1998 Feb;27(4):727-38. doi: 10.1046/j.1365-2958.1998.00718.x.
The replacement of Escherichia coli recA gene (recA[Ec]) with the Pseudomonas aeruginosa recA(Pa) gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA(Pa) protein with those of RecA(Ec) protein. Consistent with hyper-recombination activity, RecA(Pa) protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA(Pa) protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA(Ec) protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA(Pa) protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA(Pa) in comparison with RecA(Ec), RecA441 and RecA803 proteins, RecA(Pa) showed unusually high abilities: to be resistant to the displacement by SSB protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH7.5. We hypothesized that the high affinity of RecA(Pa) protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.
在大肠杆菌细胞中用铜绿假单胞菌recA（Pa）基因替换大肠杆菌recA基因（recA[Ec]），会在没有组成型SOS应答的情况下导致组成型高重组（每单位长度DNA的重组交换频率高）。为了理解这种异常体内表型的生化基础，我们在体外比较了RecA（Pa）蛋白与RecA（Ec）蛋白的重组特性。与高重组活性一致，RecA（Pa）蛋白似乎在形成联合分子方面更高效，在链交换反应中产生广泛的DNA网络，并且在与单链DNA结合（SSB）蛋白竞争单链DNA（ssDNA）结合位点方面更具优势。RecA（Pa）蛋白在体外显示出在不存在和存在（即即使在RecA（Ec）蛋白的次优条件下）SSB蛋白时，对大肠杆菌LexA阻遏物的切割能力正常（SOS调节子去阻遏的一个基本步骤）。然而，与其他高重组蛋白，如RecA441和RecA730不同，RecA（Pa）蛋白在低镁浓度下从ssDNA上置换出的SSB蛋白不足以组成型诱导SOS应答。在寻找RecA（Pa）与RecA（Ec）、RecA441和RecA803蛋白相比的特殊特征时，RecA（Pa）表现出异常高的能力：对SSB蛋白从聚（dT）上的置换具有抗性；在高盐浓度下稳定三元复合物RecA::ATP::ssDNA；以及在pH7.5时双链DNA（dsDNA）成核和dsDNA依赖性ATP水解的稳态速率都要快得多。我们推测RecA（Pa）蛋白对ssDNA，尤其是dsDNA的高亲和力是导致三元复合物结合二级DNA以启动额外重组行为而不是结合LexA阻遏物以诱导组成型SOS应答的因素。
