Structure of human Rad51 protein filament from molecular modeling and site-specific linear dichroism spectroscopy

Abstract

To get mechanistic insight into the DNA strand-exchange reaction of homologous recombination, we solved a filament structure of a human Rad51 protein, combining molecular modeling with experimental data. We build our structure on reported structures for central and N-terminal parts of pure (uncomplexed) Rad51 protein by aid of linear dichroism spectroscopy, providing angular orientations of substituted tyrosine residues of Rad51-dsDNA filaments in solution. The structure, validated by comparison with an electron microscopy density map and results from mutation analysis, is proposed to represent an active solution structure of the nucleoprotein complex. An inhomogeneously stretched double-stranded DNA fitted into the filament emphasizes the strategic positioning of 2 putative DNA-binding loops in a way that allows us speculate about their possibly distinct roles in nucleo-protein filament assembly and DNA strand-exchange reaction. The model suggests that the extension of a single-stranded DNA molecule upon binding of Rad51 is ensured by intercalation of Tyr-232 of the L1 loop, which might act as a docking tool, aligning protein monomers along the DNA strand upon filament assembly. Arg-235, also sitting on L1, is in the right position to make electrostatic contact with the phosphate backbone of the other DNA strand. The L2 loop position and its more ordered compact conformation makes us propose that this loop has another role, as a binding site for an incoming double-stranded DNA. Our filament structure and spectroscopic approach open the possibility of analyzing details along the multistep path of the strand-exchange reaction.homologous recombination ͉ HsRad51 H omologous recombination, the process of exchanging DNA strands of homologous sequences, is important for both the repair of damaged DNA and the maintenance of genomic diversity. In eukaryotes, from yeast to human, Rad51 is involved in homology search and DNA strand-exchange, central processes for recombination (1, 2). The mechanism of DNA strand-exchange appears evolutionarily conserved (3), and has been extensively studied with the bacterial RecA as a model protein (4-8). The nucleo-protein filament structures appear similar among the RecA and RecA-like proteins, including Rad51 (3, 9, 10). The first structure of a RecA filament was solved Ͼ15 years ago (11) and more recently structures of Rad51 proteins have been reported (12)(13)(14). Despite these efforts, though, no detailed filamentous structure of the human Rad51 protein has been reported and only the principal steps of the recombination process are known.RecA and Rad51 protein filaments formed in presence of DNA and ATP, or a nonhydrolysable ATP analogue, all show an extended conformation with a helical pitch of Ϸ90-100 Å. The RecA and Rad51 filament structures vary significantly depending on the presence of DNA and type of nucleotide cofactor (10,(15)(16)(17)(18)(19) suggesting that the filament structure may change during the strand-exchange reaction, possibly related to its function. Thus, a st...

A new look at the human Rad51 protein

“…In both new studies (7,8), the ATP binding site is found not in the interior of individual subunits (as suggested by earlier structural studies of the relatively inactive form of RecA) but at the subunit-subunit interface. This arrangement in active, DNA-bound recombinase filaments had been predicted by the EM image reconstructions of Egelman and colleagues (10).…”

“…In archaeal RadA and eukaryotic Rad51 proteins, this residue is highly conserved as Tyr or Phe. In both new studies (7,8), DNA binding loops in the protein interiors, inferred but largely unresolved in earlier structures, are found adjacent to the DNA, allowing the role of key amino acid residues to be assigned. The new picture of recombinase-DNA interactions, coupled to published kinetic studies (e.g., ref.…”

“…First came the appearance of a RecA protein structure that included bound DNA (7). Now comes the publication in this issue of PNAS of a model for human Rad51 protein (hsRad51) bound to DNA (8).…”

Current status and prospects of the meiosis-specific function of recombinase in plants

“…RecA-DNA 복합체의 X 선 결정구조 해석에 서 RecA는 L1 루프 및 L2 루프라고 불리는 2 개의 루프를 통 해 DNA와 결합하는 것이 밝혀졌다 (Chen et al 2008). 이 DNA 결합 루프는 RAD51에서도 보존되어 있다 (Matsuo et al, 2006;Reymer et al 2009 (Kurumizaka et al 1996. 이 C 말단 도메인은 필라 멘트의 외측에 결합한 DNA를 내측의 DNA 결합 루프로 이 끄는 게이트웨이로 간주되고 있다 (Kurumizaka et al 1996.…”

Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

“…This recombinase-ssDNA nucleoprotein filament, commonly referred to as the presynaptic filament, undergoes a conformational change upon ATP-binding such that it becomes activated for homologous DNA pairing and strand exchange (12,13). Furthermore, the presynaptic filament possesses a second binding site that can accommodate a duplex DNA molecule (14,15). As such, the presynaptic filament provides the catalytic scaffold to sample the incoming duplex DNA and form first an unstable paranemic joint with the ssDNA, which can mature into a stable plectonemic joint in which the recombining DNA strands are linked topologically (16)(17)(18)(19).…”