structural and functional analyses of five conserved positively charged residues in the l1 and n-terminal dna binding motifs of archaeal rada protein结构和功能分析的五个守恒的带正电的残留在l1和n端dna结合主题的热点rada蛋白质.pdfVIP

Structural and Functional Analyses of Five Conserved Positively Charged Residues in the L1 and N-Terminal DNA Binding Motifs of Archaeal RadA Protein 1,3 3 1,3 3 1,2,3,4 1,3 Li-Tzu Chen , Tzu-Ping Ko , Yu-Wei Chang , Kuei-An Lin , Andrew H.-J. Wang *, Ting-Fang Wang * 1 Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan, 2 Department of Life Sciences, National Taiwan University, Taipei, Taiwan, 3 Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, 4 National Core Facility of High-Throughput Protein Crystallography, Academia Sinica, Taipei, Taiwan 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 r