DNA replication, recombination, and repair processes rely on DNA helicases for local DNA strand separation. The eukaryotic Chl1 (chromosome loss 1 protein) helicase plays a role in the DNA replication stress response and is crucial for genome maintenance. However, its biochemical activity is not well-understood. In this study, we employed a single-molecule magnetic tweezers assay and ensemble biophysical methods to determine the translocation and unwinding activity of yeast Chl1 (homolog of the human ChlRl/DDX11 helicase). Our findings reveal that ATP hydrolysis is strongly coupled with translocation and unwinding, and the unwinding rate closely correlates with the rate of DNA replication. The unwinding processivity was highly dependent on force, with processive runs frequently terminated by strand-switching. In contrast to the processivity, the unwinding rate was weakly dependent on force, and the ratio of unwinding to translocation rates was close to unity, indicating that Chl1 utilizes an active helicase mechanism. As has been observed with other DNA helicases, the unwinding rate was affected by the DNA sequence, with the enzyme showing longer dwells at specific sites. Strikingly, the same sequence dependence is observable during single-stranded DNA translocation, suggesting that the sequence-specific interaction of the helicase with the translocating strand significantly contributes to the helicase's stepping mechanism. Additionally, we demonstrate that Chl1 can unwind stable G-quadruplex DNA structures that might otherwise hinder DNA replication. Our results suggest that Chl1’s unwinding activity may facilitate DNA replication by eliminating DNA secondary structures, thus aiding replication fork progression and restart to preserve genome stability.
Harami-Papp et al. (Sun,) studied this question.