Kumcu, M. E. | bioRxiv Science

bioRxiv2026

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 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=73 SRC="FIGDIR/small/705743v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@82adb8org.highwire.dtl.DTLVardef@15b1fcforg.highwire.dtl.DTLVardef@1855d10org.highwire.dtl.DTLVardef@a104ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

A screen for synthetic genetic interactions with the Saccharomyces cerevisiae hrq1ΔN allele

The Saccharomyces cerevisiae Hrq1 helicase is a functional homolog of the disease-linked human RECQL4 enzyme and has been used as a model to study RecQ4 helicase subfamily biology. Although the motor cores of Hrq1 and RECQL4 are quite similar, these proteins display distinct N-terminal domains (NTDs) of unknown function. Do these domains facilitate species-specific activities by the two helicases, or do they serve common roles despite their differences in sequence and predicted structure? We probed these questions here by analyzing an NTD-truncated isoform of Hrq1 (Hrq1{Delta}N) both in vitro and in vivo. We found that the Hrq1 NTD houses a cryptic DNA binding site and that the hrq1{Delta}N allele is distinct from both hrq1{Delta} and the catalytically inactive hrq1-K318A mutant. Using synthetic genetic array analysis of hrq1{Delta}N crossed to the yeast S. cerevisiae single-gene deletion and temperature-sensitive allele collections, we also identified hundreds of synthetic genetic interactions. As with similar analyses of hrq1{Delta} and hrq1-K318A, our results suggest roles for Hrq1 and its NTD in multiple physiological pathways that underpin genome integrity. Together, these data are guiding our ongoing efforts to understand the roles of Hrq1 and RECQL4 in genome maintenance, which will help to explain why RECQL4 mutations cause disease. ARTICLE SUMMARYThis work should interest researchers in the genome integrity and yeast disease model fields. It continues ongoing efforts to develop the Saccharomyces cerevisiae Hrq1 helicase as a model to understand the disease-linked human RECQL4 helicase. Hrq1 and RECQL4 share similar helicase cores but have divergent N-terminal domains (NTDs) of unknown function. The results demonstrate that the Hrq1 NTD contains a DNA binding site, and an HRQ1 allele lacking its NTD (hrq1{Delta}N) interacts with hundreds of mutants in defined allele collections. Thus, the hrq1{Delta}N allele is distinct from the previously characterized hrq1{Delta} and hrq1-K318A (catalytically inactive) mutants.

molecular biology↗