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Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation 1 , 2 , 3 – 4 . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor 3 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses, we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the single-stranded DNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1. A head-to-head double-ring complex of the human multifunctional DNA repair protein RAD52 mediates protection of stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor.

The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing. Cells are constantly in danger of losing or scrambling critical genetic information because of DNA damage. To cope with this stress, cells have numerous DNA repair systems. One of these systems – homology-directed DNA repair – involves the proteins BRCA1 and BRCA2, which are often missing or defective in breast and ovarian cancers. The BRCA-deficient cancer cells can still survive, but become “addicted” to other DNA repair proteins – among them a protein called RAD52. It might be possible to kill these cancer cells using drugs that stop RAD52 from working. Such treatments would have the benefit of not harming normal healthy cells, as these cells contain working BRCA proteins and can survive without RAD52. It is not currently known exactly how RAD52 allows the BRCA-deficient cells to survive, but this probably depends on RAD52’s ability to bind to single strands of DNA. Small molecules that block the interaction between the RAD52 protein and DNA might therefore help to kill cancer cells. Hengel et al. developed a high throughput biophysical method to search through a large collection of small molecules to find those that prevent RAD52 from binding to DNA. The best potential drug leads were then tested in laboratory-grown human cells and using biophysical and biochemical techniques. Computational approaches were also used to model how these molecules block the interaction between RAD52 and DNA at the atomistic level. Hengel et al. then used the information about how the small molecules bind to RAD52 to perform further computational screening. This identified a natural compound that competes with single-stranded DNA to bind to RAD52. The activity of this molecule was then validated using biophysical methods. The methods used by Hengel et al. provide the foundation for further searches for new anticancer drugs. Future studies that employ the small molecule drugs identified so far will also help to determine exactly how RAD52 works in human cells and how it helps cancer cells to survive.

One of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen’s life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks. Protein-ligand binding can induce the formation of cryptic allosteric pockets not found in the unbound protein, but predicting this computationally can be challenging. Here a combined computational and experimental workflow identifies ligands for H. pylori glutamate racemase, finding that coupled dynamics of the enzyme dimer are dampened by ligand binding.

One of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen's life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks.

Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1 . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1. Using cryo-EM, biochemical and single-molecule approaches we show that the structure of stalled DNA replication fork promotes a unique two-ring organization of human RAD52 protein which remodels the fork via DNA strand exchange.

Gilbert et al . conclude that evidence from the Open Science Collaboration’s Reproducibility Project: Psychology indicates high reproducibility, given the study methodology. Their very optimistic assessment is limited by statistical misconceptions and by causal inferences from selectively interpreted, correlational data. Using the Reproducibility Project: Psychology data, both optimistic and pessimistic conclusions about reproducibility are possible, and neither are yet warranted.