Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation to clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 cystic fibrosis patients in registries and clinics in North America and Europe. Among these patients, 159 CFTR variants had an allele frequency of ≥0.01%. These variants were evaluated for both clinical severity and functional consequence with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of cystic fibrosis patients enabled assignment of 12 of the remaining 32 variants as neutral while the other 20 variants remained indeterminate. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically-relevant genomic variation.

SUMMARY Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ~50 monomers per second. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments, and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. Based upon these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.

Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double-strand break (DSB) repair, and the meiosis-specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis-specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real-time single-molecule imaging to probe how the Hed1- and Rad54-mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.