Conformations of Three-Stranded DNA Structures Formed in Presence and in Absence of the RecA Protein

Abstract

Using FTIR and UV spectroscopies, we have studied the structures of three-stranded DNA complexes (TSC) having two identical strands, containing all four bases, in parallel orientation. In the first system, an intermolecular TSC is formed by the addition of the third strand (ssDNA) previously coated with RecA protein to an hairpin duplex (dsDNA), in presence of ATP gamma S. In the second one, the formation of an intramolecular triplex is forced by folding back twice on itself an oligonucleotide. The sequences of the three strands are the same in both systems. The formation of the RecA-TSC, which accommodates all four bases, is evidenced by gel retardation assay, and by its biphasic melting profile observed by UV spectroscopy. Using FTIR spectroscopy, N-type sugars are detected in this structure. This shows that in the RecA-TSC studied in presence of the protein, the nucleic acid part adopts an extended form, in agreement with the model proposed by Zhurkin et al. (1,2) and electron microscopy observations (3-6). In contrast, the RecA-free intramolecular triplex in a non extended form has S-type sugars.

DNA stretching and compression: large-scale simulations of double helical structures 1 1Edited by I. Tinoco

“…Earlier, one of us has predicted a B-to-A-like transition in DNA stretched under the restrictions imposed by the geometry of the RecA-DNA ®lament in the so-called``extended R-form'' (Zhurkin et al, 1994a,b). This transition, subsequently con-®rmed by the partial C2 H -endo to C3 H -endo switching of sugar puckering in FTIR and NMR studies of DNA-RecA complexes (Dagneaux et al, 1995;Nishinaka et al, 1997Nishinaka et al, , 1998, also agrees with the present study: A 1 -forms are favored but B 1 -forms are disfavored at $5 A Ê /bp stretching (Figure 1(a) and (b)). Like the earlier extended R-form model (Zhurkin et al, 1994b), the DNA stretched here has an increased hydrophobic surface compared to canonical A and B-form DNA.…”

“…Such advantageous interactions are well known in the DNA crystallographic literature (Wahl et al, 1996;Mandel-Gutfreund et al, 1998). Geometrical analyses further show that this sugarsugar contact is more favorable when the sugar pucker is C3 H -endo (in accordance with FTIR measurements (Dagneaux et al, 1995)), and the sugars are deoxyriboses (in accordance with the low af®nity of RecA for RNA (McEntee et al, 1981)).…”

“…We assume that our`h igh salt'' models more closely mimic the environmental conditions in the center of the RecA ®lament, where there are numerous positively charged amino acids, especially in the vicinity of the L2 loop (Story et al, 1992). We further choose the A-like (A 1 ) over the B-like (B t ) form in the recombination model detailed below, given that a C2 H -endo to C3 H -endo conformational transition is detected in FTIR measurements of the RecA-threestranded complex (Dagneaux et al, 1995). The reasoning behind our protein-DNA model, however, is applicable to any of the aforementioned conformations: A 1 , A t , B 1 and B t , all of which share similar global features.…”

A Molecular Model for RecA-Promoted Strand Exchange via Parallel Triple-Stranded Helices

“…The evolution of the sugar puckers of the major-groove triplex, presented in Fig. 5 c (the same is true for the minor-groove triplex, data not shown), agrees with the results of a Fourier transform infrared study on RecA-complexed triplexes, probably following strand exchange (Dagneaux et al, 1995a). This study detected sugars in a north conformation (C3Ј-endo) in the extended RecA-complexed form, but not in the relaxed conformation.…”

Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: A model for homology search in homologous genetic recombination

“…In a previous model-building study, Zhurkin et al (48) presented a triplex DNA structure in which sugar puckers were in the N-type but did not take into account the deoxyribose-base stacking revealed by our recent NMR study. A FTIR spectroscopic study also suggested that a triplex DNA in an active RecA-nucleoprotein filament had the N-type sugar conformation (75). The model proposed by Zhurkin et al (48) postulated that all three bases were hydrogen bonded to each other, placing constraints on the formation of the triple helix.…”