Abstract
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 Å 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.
Highlights
The RecA family of DNA strand exchange proteins exists in all three kingdoms of life
Structural and biochemical analyses in this study indicate that these five basic residues play key roles in DNA binding and strand exchange activities (Figures 2–7)
In the 31 overwound right-handed helical filament of Sulfolobus solfataricus RadA (SsoRadA) proteins, these five basic residues generate a positively charged surface for a palm structure formed by the L1 motif and the N-terminal domain (NTD)
Summary
The RecA family of DNA strand exchange proteins exists in all three kingdoms of life. Members of the RecA protein family include prokaryotic RecA, archaeal RadA and Rad, and eukaryotic Rad and Dmc. Members of the RecA protein family include prokaryotic RecA, archaeal RadA and Rad, and eukaryotic Rad and Dmc1 These proteins play a central role in homologous recombination, an error-free DNA repair mechanism [for reviews see 1, 2, 3]. While RecA-deficient E. coli cells or Rad51-deficient yeast cells are viable, Rad51-deficient vertebrate cells are not. The latter accumulate chromosomal breaks prior to cell death [4]. Rad and its meiosis-specific homolog, Dmc, are indispensable for meiosis [5,6]. RecA family proteins have important roles in cell proliferation, genome maintenance, and genetic diversity, in higher eukaryotes
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