Base Orientation of Second DNA in RecA·DNA Filaments

Abstract

To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA⅐DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(d⑀A) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands. The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA⅐DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.The RecA protein plays a crucial role in homologous recombination in Escherichia coli: it regulates the synthesis of proteins involved in recombination reaction, including RecA itself, and catalyzes strand-exchange reaction (1). RecA can, in the presence of cofactor ATP, promote pairing of two complementary DNA strands (2) and also perform strand exchange between two homologous DNA molecules in vitro (3, 4). In these reactions, RecA first binds with high cooperativity to a singlestranded DNA (5) forming a nucleoprotein filament (6). In the complex, the protein monomers are arranged in a helical manner around the DNA, and the DNA is elongated about 50% in length. This RecA single-stranded DNA nucleofilament can then bind a second DNA (either single-or double-stranded DNA) and pair complementary parts.From fluorescence measurements of various probes attached to the DNA bases, we have concluded that direct base-base interaction of the Watson-Crick type contributes to the sequence recognition by RecA (7, 8). Base-base interaction can occur between all three DNA strands in the RecA⅐DNA complex, supporting formation of a triplex DNA structure, as has been suggested by other studies (9, 10). Several models of different base-triads, involved in triplex-structure of recombination intermediates have been proposed (11,12) In order to investigate the base-base interactions and understand more about the mechanism of recognition between complementary DNA strands in RecA, we earlier assessed the orientation (roll and tilt angles) of DNA bases in RecA⅐DNA complexes by linear dichroism (LD) 1 spectroscopy combined with small-angle neutron scattering (SANS) and chromophorereplacement studies (13-15). LD measures, on an aligned sample, the absorption difference of light polarized parallel a...

Modeling the Homologous Recombination Process: Methods, Successes and Challenges

“…Interestingly, this group showed that pulling the 3 extremities unwinds the double helix; when reaching 50% extension, the DNA unwinding reaches exactly the 40% value found in the RecA filaments [72]. In addition, at 50% extension the base pairs become perpendicular to the DNA axis, in agreement to the experimental results of Nordén and Takahashi [73,74], while beyond and above the 50% value, the bases tilt in order to conserve part of their stacking interaction; switching to a perpendicular orientation permits to optimally regain the lost stacking interaction within groups of stacked bases, at the expense of introducing large intercalation sites in the filament every three base pairs [75]. Indeed, the structure resulting from their simulation in the late 1990s is strikingly similar to the structure of DNA bound to the filament, solved by crystallography ten years later [72,75,76].…”

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

“…We have previously used LD to study the DNA base orientation in the complex of DNA and RecA protein, which plays a crucial role in the DNA repair in E. coli (5,6). RecA regulates expression and activity of DNA repair enzymes by its coprotease activity for the LexA repressor and facilitates DNA repair by catalyzing the strand exchange between homologous DNA strands (7)(8)(9).…”

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction

“…Further LD studies showed that the bases of the dsDNA bound to the second site of the recombinase filament were perpendicular to the filament axis. 22 Thus, the activated recombinase filament facilitates the pair formation between DNA bases of ssDNA and dsDNA. The kinetic studies showed that Ca 2+ and Swi-Sfr1 accelerate the new dsDNA formation step.…”