DNA binding contributes to dominant-negative effects of a catalytically inactive RecQ4-family helicase.

DNA inter-strand crosslinks (ICLs) are highly cytotoxic lesions that require coordinated processing for repair. The RecQ4-family helicase Hrq1 promotes ICL repair in Saccharomyces cerevisiae, yet the mechanistic basis of its function remains unclear. Notably, the catalytically inactive hrq1-K318A allele confers greater sensitivity to ICL-inducing agents than deletion of HRQ1, suggesting a dominant-negative effect. To define the basis of this phenotype, we performed a genetic suppressor screen combined with biochemical and structural analyses. Spontaneous suppressors of hrq1-K318A sensitivity were overwhelmingly intragenic second-site mutations, many of which are predicted to destabilize the protein or impair its ability to bind DNA. In all cases, these mutations alleviated the dominant-negative repair defect. Biochemical characterization of representative mutants, including a rationally designed DNA-binding mutant, demonstrated that disruption of DNA binding suppresses hrq1-K318A toxicity even when protein stability is retained. These findings support a model in which DNA engagement by a catalytically inactive RecQ4-family helicase contributes to dominant-negative interference with DNA repair. More broadly, this work provides insight into how incomplete loss-of-function alleles of human RECQL4 may disrupt genome maintenance pathways.