Solution formation of Holliday junctions in inverted-repeat DNA sequences

Abstract

The structure of Holliday junctions has now been well characterized at the atomic level through single-crystal X-ray diffraction in symmetric (inverted-repeat) DNA sequences. At issue, however, is whether the formation of these four-stranded complexes in solution is truly sequence dependent in the manner proposed or is an artifact of the crystallization process and, therefore, has no relevance to the behavior of this central intermediate in homologous recombination and recombination-dependent cellular processes. Here, we apply analytical ultracentrifugation to demonstrate that the sequence d(CCGGTACCGG), which crystallizes in the stacked-X form of the junction, assembles into four-stranded junctions in solution in a manner that is dependent on the DNA and cation concentrations, with an equilibrium established between the junction and duplex forms at 100-200 microM DNA duplex. In contrast, the sequence d(CCGCTAGCGG), which has been crystallized as B-DNA, is seen to adopt only the double-helical form at all DNA and salt concentrations that were tested. Thus, the ACC trinucleotide core is now shown to be important for the formation of Holliday junctions in both crystals and in solution and can be estimated to contribute approximately -4 kcal/mol to stabilizing this recombination intermediate in inverted-repeat sequences.

Figure 1

Holliday junctions. Schematics of the open-X (a) and stacked-X (b) junctions. (c) Model of the stacked-X junction derived from anomalous gel migration studies of immobilized junctions (5) showing the angle relating the helical axes of the stacked duplex arms [J_twist (19)]. (d) Single-crystal structure of the inverted-repeat sequence d(CCGGTACCGG) (8), with the 4 bp/6 bp arrangement of stacked arms.

Figure 2

Analytical ultracentrifugation integral distribution plots for d(CCGGTACCGG) in 25 mM sodium cacodylate buffer at pH 7.0 and 20 °C, with varied DNA and CaCl₂ concentrations (as labeled). The sedimentation coefficients calculated from the B-DNA structure of d(CCGCTAGCGG) (8), from the DNA coordinates of the open-X junction in the Cre–lox complex (28) (with average 5 bp extended arms), and from the stacked-X junction structure of d(CCGGTACCGG) (8) are labeled. The hashed region indicates the range of coefficients for models of the stacked-X junction with different angles relating the stacked duplex arms (2.44 S for J_twist = 40°, 2.42 S for J_twist = 60°, and 2.45 S for J_twist = 20°, with 4 bp/6 bp stacked arms; see Figure 1d) and with different arrangements of stacked arms (2.44 S for 6 bp/4 bp and 2.48 S for 5 bp/5 bp, and J_twist = 40°). Differences between the calculated and experimental S values may reflect systematic errors from the HYDROPRO program not accounting for flexibility in or cation condensation onto the DNA molecules.