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...
Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.
Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca2+ than of Mg2+, we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca2+ induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg2+, ADP/Mg2+ or ADP/Ca2+ does not. A high strand exchange activity is observed for the filament formed with ATP/Ca2+, whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca2+ stabilizes the loop conformation and thereby the protein–DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.
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