Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
PLoS One. 2007 Sep 12;2(9):e858. doi: 10.1371/journal.pone.0000858.
RecA family proteins engage in an ATP-dependent DNA strand exchange reaction that includes a ssDNA nucleoprotein helical filament and a homologous dsDNA sequence. In spite of more than 20 years of efforts, the molecular mechanism of homology pairing and strand exchange is still not fully understood. Here we report a crystal structure of Sulfolobus solfataricus RadA overwound right-handed filament with three monomers per helical pitch. This structure reveals conformational details of the first ssDNA binding disordered loop (denoted L1 motif) and the dsDNA binding N-terminal domain (NTD). L1 and NTD together form an outwardly open palm structure on the outer surface of the helical filament. Inside this palm structure, five conserved basic amino acid residues (K27, K60, R117, R223 and R229) surround a 25 A pocket that is wide enough to accommodate anionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that these five positively charged residues are essential for DNA binding and for RadA-catalyzed D-loop formation. We suggest that the overwound right-handed RadA filament represents a functional conformation in the homology search and pairing reaction. A new structural model is proposed for the homologous interactions between a RadA-ssDNA nucleoprotein filament and its dsDNA target.
RecA 家族蛋白参与 ATP 依赖性 DNA 链交换反应，其中包括 ssDNA 核蛋白螺旋丝和同源双链 DNA 序列。尽管经过 20 多年的努力，同源配对和链交换的分子机制仍未完全理解。在这里，我们报告了 Sulfolobus solfataricus RadA 过度缠绕右手螺旋丝的晶体结构，每个螺旋螺距有三个单体。该结构揭示了第一个 ssDNA 结合无序环（表示为 L1 基序）和 dsDNA 结合 N 端结构域（NTD）的构象细节。L1 和 NTD 共同在螺旋丝的外表面形成向外张开的手掌结构。在这个手掌结构内，五个保守的碱性氨基酸残基（K27、K60、R117、R223 和 R229）包围一个 25A 的口袋，足以容纳阴离子 ssDNA、dsDNA 或两者。生化分析表明，这五个带正电荷的残基对于 DNA 结合和 RadA 催化的 D 环形成是必不可少的。我们认为，过度缠绕的右手 RadA 丝代表了同源搜索和配对反应中的功能构象。提出了一个新的结构模型，用于 RadA-ssDNA 核蛋白丝与其 dsDNA 靶标的同源相互作用。
