Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

Abstract

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.

Junction resolving enzymes use multivalency to keep the Holliday junction dynamic

“…15d). It has been shown that a nick does not prevent branch migration 37 . hMus81-Eme1 efficiently cleaved n-J5m but not J5m (Supplementary Fig.…”

“…22-bp dsDNA was annealed by mixing the two strands with the molar ratio 1:1 (final concentration ~10 μM each) in 10 mM Tris:HCl (pH 8.0) and 50 mM NaCl followed by slow cooling from 90°C to room temperature for ~ 2 hours. J5m, n-J5m and J0m were constructed as previously described 7,37,45 . When J5m undergoes spontaneous branch migration, there are six possible donor-acceptor separations of 10, 12, 14, 16, 18, 20 bp for different branch positions.…”

Active Control of Repetitive Structural Transitions between Replication Forks and Holliday Junctions by Werner Syndrome Helicase

“…Single-stranded breaks are a ubiquitous defect of the DNA helix. It has been shown that even a single heterologous base pair is able to block the spontaneous branch migration of a four-way junction (Biswas et al, 1998), whereas single-stranded breaks do not hinder spontaneous branch migration (Palets et al, 2010). Branch migration catalyzed by T7 gp4 was stalled by a single heterologous base pair (Rasnik et al, 2008).…”

Implications of Metastable Nicks and Nicked Holliday Junctions in Processing Joint Molecules in Mitosis and Meiosis

“…The nHJ is probably metastable as it occupies a local thermodynamic minimum. However, isomerization into a cHJ through branch migration is possible in solution [ 49 ], and it is, therefore, likely in vivo as well (spontaneously or enzymatically driven). Interestingly, the nick that is left behind after branch migration should ameliorate the need for the concerted action of topoisomerases as they enable strand swiveling during branch migration ( Figure 3 D).…”