Witte Gregor, Fedorov Roman, Curth Ute
Institute for Biophysical Chemistry, Hannover Medical School, D-30625 Hanover, Germany.
Biophys J. 2008 Mar 15;94(6):2269-79. doi: 10.1529/biophysj.107.121533. Epub 2007 Dec 7.
Due to their involvement in processes such as DNA replication, repair, and recombination, bacterial single-stranded DNA binding (SSB) proteins are essential for the survival of the bacterial cell. Whereas most bacterial SSB proteins form homotetramers in solution, dimeric SSB proteins were recently discovered in the Thermus/Deinococcus group. In this work we characterize the biophysical properties of the SSB protein from Thermus aquaticus (TaqSSB), which is structurally quite similar to the tetrameric SSB protein from Escherichia coli (EcoSSB). The binding of TaqSSB and EcoSSB to single-stranded nucleic acids was found to be very similar in affinity and kinetics. Mediated by its highly conserved C-terminal region, TaqSSB interacts with the chi-subunit of E. coli DNA polymerase III with an affinity that is similar to that of EcoSSB. Using analytical ultracentrifugation, we show that TaqSSB mutants are able to form tetramers in solution via arginine-mediated hydrogen-bond interactions that we identified in the crystal packing of wild-type TaqSSB. In EcoSSB, we identified a homologous arginine residue involved in the formation of higher aggregates and metastable highly cooperative single-stranded DNA binding under low salt conditions.
由于参与DNA复制、修复和重组等过程，细菌单链DNA结合（SSB）蛋白对于细菌细胞的存活至关重要。虽然大多数细菌SSB蛋白在溶液中形成同四聚体，但最近在嗜热栖热菌/异常球菌类群中发现了二聚体SSB蛋白。在这项工作中，我们表征了嗜热水生栖热菌（TaqSSB）的SSB蛋白的生物物理特性，其结构与大肠杆菌（EcoSSB）的四聚体SSB蛋白非常相似。发现TaqSSB和EcoSSB与单链核酸的结合在亲和力和动力学方面非常相似。通过其高度保守的C末端区域介导，TaqSSB与大肠杆菌DNA聚合酶III的χ亚基相互作用，其亲和力与EcoSSB相似。使用分析超速离心，我们表明TaqSSB突变体能够通过我们在野生型TaqSSB的晶体堆积中鉴定出的精氨酸介导的氢键相互作用在溶液中形成四聚体。在EcoSSB中，我们鉴定出一个同源精氨酸残基，其参与低盐条件下更高聚集体的形成和亚稳态高度协同的单链DNA结合。
