Waldman Vincent M, Weiland Elizabeth, Kozlov Alexander G, Lohman Timothy M
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, 660 S. Euclid Avenue, Box 8231, 63110-1093, USA
Nucleic Acids Res. 2016 May 19;44(9):4317-29. doi: 10.1093/nar/gkw262. Epub 2016 Apr 15.
Escherichia coli single-stranded DNA binding protein (SSB) is an essential homotetramer that binds ssDNA and recruits multiple proteins to their sites of action during genomic maintenance. Each SSB subunit contains an N-terminal globular oligonucleotide/oligosaccharide binding fold (OB-fold) and an intrinsically disordered C-terminal domain. SSB binds ssDNA in multiple modes in vitro, including the fully wrapped (SSB)65 and (SSB)56 modes, in which ssDNA contacts all four OB-folds, and the highly cooperative (SSB)35 mode, in which ssDNA contacts an average of only two OB-folds. These modes can both be populated under physiological conditions. While these different modes might be used for different functions, this has been difficult to assess. Here we used a dimeric SSB construct with two covalently linked OB-folds to disable ssDNA binding in two of the four OB-folds thus preventing formation of fully wrapped DNA complexes in vitro, although they retain a wild-type-like, salt-dependent shift in cooperative binding to ssDNA. These variants complement wild-type SSB in vivo indicating that a fully wrapped mode is not essential for function. These results do not preclude a normal function for a fully wrapped mode, but do indicate that E. coli tolerates some flexibility with regards to its SSB binding modes.
大肠杆菌单链DNA结合蛋白（SSB）是一种必需的同四聚体，在基因组维持过程中与单链DNA结合，并将多种蛋白质招募到其作用位点。每个SSB亚基包含一个N端球状寡核苷酸/寡糖结合折叠（OB折叠）和一个内在无序的C端结构域。SSB在体外以多种模式结合单链DNA，包括完全包裹的（SSB）65和（SSB）56模式，其中单链DNA与所有四个OB折叠接触，以及高度协同的（SSB）35模式，其中单链DNA平均仅与两个OB折叠接触。这些模式在生理条件下都可能出现。虽然这些不同的模式可能用于不同的功能，但这很难评估。在这里，我们使用了一种具有两个共价连接的OB折叠的二聚体SSB构建体，以禁用四个OB折叠中的两个中的单链DNA结合，从而防止在体外形成完全包裹的DNA复合物，尽管它们在与单链DNA的协同结合中保留了类似野生型的盐依赖性转变。这些变体在体内补充野生型SSB，表明完全包裹模式对功能不是必需的。这些结果并不排除完全包裹模式的正常功能，但确实表明大肠杆菌在其SSB结合模式方面具有一定的灵活性。
