Effect of Single-Strand Break on Holliday Junction Migration Dynamics: A Single-Molecule Fluorescence Study

Single-strand breaks (SSBs) are the most common DNA lesions in living cells. SSBs occur three orders of magnitude more frequently than double-strand breaks. Unrepaired SSBs lead to blockage or collapse of DNA replication forks, possibly causing formation of double-strand breaks. Holliday junctions (HJ) play central role in various DNA functions including repair of lesions, replication, homologous and site-specific recombination. Branch migration, either spontaneous or protein-mediated, is among widely employed mechanisms in these functions. Therefore, elucidation of the SSB effect on spontaneous branch migration of HJs is a problem of great importance. To accomplish this task, we employed single-molecule FRET approach developed before, allowing us to follow spontaneous branch migration of one HJ at a single base pair level in real time. One SSB was incorporated in the middle of the homologous region of mobile HJ with the donor and acceptor dyes placed on its opposite arms. The data showed that branch migration does not stop at these lesions or reflects from them. Our previous results showed that branch migration is a step-wise process and one step can cover entire homology region. The analysis of the time trajectories showed that such long steps are not prevented by SSBs. Step size distributions for HJs containing SSB were obtained and such characteristics of these distributions as the mean hop size and the distributions width remain virtually the same as in the designs without a break. Lifetimes of folded states were found to be very similar to that in control, just slightly increasing around the SSB in the AT-rich design. We also studied an immobile HJ with a SSB at the junction. Its arms remain in antiparallel orientation, very similarly to our previous studies.Supported by the NSF grant PHY-0615590 (YLL).