DNA binding converts an inactive RecQ4-family helicase into a dominant-negative DNA repair factor

DNA inter-strand crosslinks (ICLs) are highly cytotoxic lesions that require coordinated processing to faithfully repair. Members of the RecQ4 helicase family, such as <i>Saccharomyces cerevisiae</i> Hrq1, are implicated in ICL repair yet remain poorly understood mechanistically. Hrq1 promotes ICL repair by stimulating the nuclease Pso2, but cells expressing a helicase-dead <i>hrq1-K318A</i> allele are more sensitive to ICL damage than <i>hrq1Δ</i> cells, indicating a dominant-negative defect. To define the basis of this toxicity, we used unbiased genetic suppressor screening, structural modeling, and biochemical analyses. Spontaneous suppressors of <i>hrq1-K318A</i> sensitivity were overwhelmingly intragenic second-site mutations in <i>hrq1</i>that either destabilized Hrq1-K318A or disrupted its ability to bind DNA. In all cases, these mutations relieved the dominant-negative repair defect. Biochemical characterization of representative mutants, including a rationally designed DNA-binding mutant, demonstrated that loss of DNA binding abolishes helicase activity and suppresses Hrq1-K318A toxicity despite retaining protein stability. Together, these findings identify persistent DNA binding by a catalytically inactive RecQ4-family helicase as the central driver of dominant-negative ICL repair defects and provide mechanistic insight into how incomplete loss-of-function alleles of human RECQL4 may actively interfere with genome maintenance pathways.