Alexseyev A A, Baitin D M, Kuramitsu S, Ogawa T, Ogawa H, Lanzov V A
Department of Molecular and Radiation Biophysics, Petersburg Nuclear Physics Institute, Russian Academy of Sciences, Gatchina/St. Petersburg, Russia.
Mol Microbiol. 1997 Jan;23(2):255-65. doi: 10.1046/j.1365-2958.1997.1961557.x.
RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the C-terminal subdomain of the protein. RecA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42 degrees C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecA-mut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.
RecA2278 - 5是一种突变型RecA蛋白（RecAmut），在该蛋白C末端亚结构域的α - 螺旋H中有两个氨基酸替换，即甘氨酸278被苏氨酸取代以及缬氨酸275被苯丙氨酸取代。RecA2278 - 5突变细胞的不同寻常之处在于它们对重组具有温度敏感性，但对紫外线损伤的DNA修复和SOS反应几乎正常。对纯化的RecAmut蛋白进行生化分析发现，其温度敏感性可通过该蛋白与配体的预先结合而受到抑制。事实上，在无ATP的情况下，将RecAmut蛋白在限制温度（42℃）下预热几分钟，会导致在42℃时与同源重组相关的许多活性受到抑制，包括单链和双链DNA结合、对ATP的高亲和力结合、单链或双链DNA依赖性ATP酶、RecA - RecA相互作用以及链转移能力。在相同条件下，二元复合物RecAmut::ATP仅在两种活性上有所下降，即双链DNA结合和对ATP的高亲和力结合。除了ATP外，通过其无DNA的ATP酶活性的恢复判断，乙酸钠（1.5 M）被证明是另一个可在42℃稳定RecAmut蛋白的因素。高盐（通过其非特异性结合）和ATP（特异性结合）对表观蛋白质折叠稳定性影响的相似性表明，RecA C末端结构域的结构稳定性是RecA蛋白与ATP在RecA::ATP::ssDNA突触前复合物形成过程中正确相互作用的条件之一。对ATP亲和力的降低被认为是决定RecA突变蛋白特定重组（而非修复）温度敏感性的因素。最后，我们表明C末端结构域的稳定性对于该蛋白的双链DNA结合活性似乎是必要的。
