Mechanistic Study of ATP-Dependent Regulation of the Hrq1 Oligomeric State and Its DNA Unwinding Activity in Saccharomyces cerevisiae.

The yeast Hrq1 protein is an important model for studying the human RECQL4 helicase and RECQL4-associated diseases. In this study, we simultaneously isolated and characterized two stable oligomeric states of Hrq1 expressed in insect cells: a monomeric form (Hrq1mon) and a higher-order oligomeric fraction (Hrq1-oligo). Intriguingly, ATP not only promotes the dissociation of the oligomeric fraction into monomeric or smaller Hrq1-containing species but also markedly stimulates the DNA-unwinding activities of both forms. Furthermore, Hrq1-mediated unwinding is strictly dependent on a forked DNA substrate, and its activity increases with increasing lengths of either the 5'- or 3'-single-stranded DNA tail. Based on the limited unwinding processivity of Hrq1, we propose a "relay-style" mechanism in which an initial Hrq1 monomer partially unwinds the duplex and subsequently dissociates, allowing additional Hrq1 monomers to bind the newly exposed 3' single-stranded region and continue the unwinding reaction. However, this process can be inhibited by ScRPA through competition for the exposed single-stranded DNA. These findings provide new insights into the oligomeric-state-dependent regulation and unwinding mechanism of Hrq1 and may contribute to a broader understanding of RecQ family helicases.