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 Saccharomyces cerevisiae 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 hrq1-K318A allele are more sensitive to ICL damage than hrq1Δ 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 hrq1-K318A sensitivity were overwhelmingly intragenic second-site mutations in hrq1that 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.