Biology subjects

Simmons, R. H.

Publications and source records attributed to Simmons, R. H..

3 recordsLinked to original sources

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

Dimerization of Cdc13 is essential for dynamic DNA exchange on telomeric DNA

Single-stranded DNA (ssDNA) binding proteins (ssBPs) are essential in eukaryotes to protect telomeres from nuclease activity. In Saccharomyces cerevisiae, the ssBP Cdc13 is an essential protein that acts as a central regulator of telomere length homeostasis and chromosome end protection, both alone and as part of the Cdc13-Stn1-Ten1 (CST) complex. Cdc13 has high binding affinity for telomeric ssDNA, with a very slow off-rate. Previously, we reported that despite this tight ssDNA binding, Cdc13 rapidly exchanges between bound and unbound telomeric ssDNA substrates, even at sub-stoichiometric concentrations of competitor ssDNA. This dynamic DNA exchange (DDE) is dependent on the presence and length of telomeric repeat sequence ssDNA and requires both Cdc13 DNA binding domains, OB1 and OB3. Here we investigated if Cdc13 dimerization is important for DDE by characterizing the dimerization mutant Cdc13-L91R. Using mass photometry, we confirmed that Cdc13-L91R fails to dimerize in solution, even in the presence of ssDNA. Gel-based DDE assays revealed that Cdc13-L91R fails to undergo ssDNA exchange compared to recombinant wild-type protein. Biolayer interferometry demonstrated that this effect was not due to differences in ssDNA binding kinetics. Thus, dimerization of Cdc13 is essential for DDE, and we model how this may impact telomere biology in vivo. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/645294v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18f34fdorg.highwire.dtl.DTLVardef@e00e71org.highwire.dtl.DTLVardef@1d4400aorg.highwire.dtl.DTLVardef@1b87a7e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA

Telomerase is the enzyme that lengthens telomeres and is tightly regulated by a variety of means to maintain genome integrity. Several DNA helicases function at telomeres, and we previously found that the Saccharomyces cerevisiae helicases Hrq1 and Pif1 directly regulate telomerase. To extend these findings, we are investigating the interplay between helicases, single-stranded DNA (ssDNA) binding proteins (ssBPs), and telomerase. The yeast ssBPs Cdc13 and RPA differentially affect Hrq1 and Pif1 helicase activity, and experiments to measure helicase disruption of Cdc13/ssDNA complexes instead revealed that Cdc13 can exchange between substrates. Although other ssBPs display dynamic binding, this was unexpected with Cdc13 due to the reported in vitro stability of the Cdc13/telomeric ssDNA complex. We found that the DNA exchange by Cdc13 occurs rapidly at physiological temperatures, requires telomeric repeat sequence DNA, and is affected by ssDNA length. Cdc13 truncations revealed that the low-affinity binding site (OB1), which is distal from the high-affinity binding site (OB3), is required for this intermolecular dynamic DNA exchange (DDE). We hypothesize that DDE by Cdc13 is the basis for how Cdc13 "moves" at telomeres to alternate between modes where it regulates telomerase activity and assists in telomere replication.

biochemistry↗