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RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52–ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23–1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3–8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

Many postdoctoral fellows and scholars who hope to secure tenure-track faculty positions in the United States apply to the National Institutes of Health (NIH) for a Pathway to Independence Award. This award has two phases (K99 and R00) and provides funding for up to five years. Using NIH data for the period 2006-2022, we report that ∼230 K99 awards were made every year, ∼40% of K99 awardees were women, and that ∼85% of K99 awardees went on to receive an R00 award. Institutions with the most NIH funding produced the most recipients of K99 awards and recruited the most recipients of R00 awards. The time between a researcher starting an R00 award and receiving a major NIH award (such as an R01) ranged between 4.6 and 7.4 years, and was significantly longer for women, for those who remained at their home institution, and for those hired by an institution that was not one of the 25 institutions with the most NIH funding. Shockingly, there has yet to be a K99 awardee at a historically Black college or university. We go on to show how K99 awardees flow to faculty positions, and to identify various factors that influence the future success of individual researchers and, therefore, also influence the composition of biomedical faculty at universities in the US.

The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as another interacting partner of DSS1 and shows that both proteins are important players in the SSA and BIR pathways of DSB repair.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

Accurate DNA repair and replication are critical for genomic stability and cancer prevention. RAD51 and its gene family are key regulators of DNA fidelity through diverse roles in double-strand break repair, replication stress, and meiosis. RAD51 is an ATPase that forms a nucleoprotein filament on single-stranded DNA. RAD51 has the function of finding and invading homologous DNA sequences to enable accurate and timely DNA repair. Its paralogs, which arose from ancient gene duplications of RAD51, have evolved to regulate and promote RAD51 function. Underscoring its importance, misregulation of RAD51, and its paralogs, is associated with diseases such as cancer and Fanconi anemia. In this review, we focus on the mammalian RAD51 structure and function and highlight the use of model systems to enable mechanistic understanding of RAD51 cellular roles. We also discuss how misregulation of the RAD51 gene family members contributes to disease and consider new approaches to pharmacologically inhibit RAD51.

To maintain stable genomes and to avoid cancer and aging, cells need to repair a multitude of deleterious DNA lesions, which arise constantly in every cell. Processes that support genome integrity in normal cells, however, allow cancer cells to develop resistance to radiation and DNA damaging chemotherapeutics. Chemical inhibition of the key DNA repair proteins and pharmacologically induced synthetic lethality have become instrumental in both dissecting the complex DNA repair networks and as promising anticancer agents. The difficulty in capitalizing on synthetically lethal interactions in cancer cells is that many potential targets do not possess well-defined small molecule binding determinates. In this review we will discuss several successful campaigns to identify and leverage small-molecule inhibitors of the DNA repair proteins, from PARP1, a paradigm case for clinically successful small molecule inhibitors, to coveted new targets, such as RAD51 recombinase, RAD52 DNA repair protein, MRE11 nuclease and WRN DNA helicase.

Conformational dynamics has an established role in enzyme catalysis, but its contribution to ligand binding and specificity is largely unexplored. Here we used the Tiam1 PDZ domain and an engineered variant (QM PDZ) with broadened specificity to investigate the role of structure and conformational dynamics in molecular recognition. Crystal structures of the QM PDZ domain both free and bound to ligands showed structural features central to binding (enthalpy), while nuclear-magnetic-resonance-based methyl relaxation experiments and isothermal titration calorimetry revealed that conformational entropy contributes to affinity. In addition to motions relevant to thermodynamics, slower microsecond to millisecond switching was prevalent in the QM PDZ ligand-binding site consistent with a role in ligand specificity. Our data indicate that conformational dynamics plays distinct and fundamental roles in tuning the affinity (conformational entropy) and specificity (excited-state conformations) of molecular interactions. More broadly, our results have important implications for the evolution, regulation, and design of protein-ligand interactions.

Accurate DNA replication is essential for genomic stability and cancer prevention. Homologous recombination is important for high-fidelity DNA damage tolerance during replication. How the homologous recombination machinery is recruited to replication intermediates is unknown. Here, we provide evidence that a Rad51 paralog-containing complex, the budding yeast Shu complex, directly recognizes and enables tolerance of predominantly lagging strand abasic sites. We show that the Shu complex becomes chromatin associated when cells accumulate abasic sites during S phase. We also demonstrate that purified recombinant Shu complex recognizes an abasic analog on a double-flap substrate, which prevents AP endonuclease activity and endonuclease-induced double-strand break formation. Shu complex DNA binding mutants are sensitive to methyl methanesulfonate, are not chromatin enriched, and exhibit increased mutation rates. We propose a role for the Shu complex in recognizing abasic sites at replication intermediates, where it recruits the homologous recombination machinery to mediate strand specific damage tolerance., The homologous recombination machinery needs to be recruited at replication intermediates for accurate functioning. Here, the authors reveal that a Rad51 paralog-containing complex, called the Shu complex, recognizes and enables tolerance of predominantly lagging strand abasic sites.

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. DOI: http://dx.doi.org/10.7554/eLife.14740.001, eLife digest 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. DOI: http://dx.doi.org/10.7554/eLife.14740.002

High fidelity homologous DNA recombination depends on mismatch repair (MMR), which antagonizes recombination between divergent sequences by rejecting heteroduplex DNA containing excessive nucleotide mismatches. The hMSH2-hMSH6 heterodimer is the first responder in postreplicative MMR and also plays a prominent role in heteroduplex rejection. Whether a similar molecular mechanism underlies its function in these two processes remains enigmatic. We have determined that hMSH2-hMSH6 efficiently recognizes mismatches within a D-loop recombination initiation intermediate. Mismatch recognition by hMSH2-hMSH6 is not abrogated by human replication protein A (HsRPA) bound to the displaced single-stranded DNA (ssDNA) or by HsRAD51. In addition, ATP-bound hMSH2-hMSH6 sliding clamps that are essential for downstream MMR processes are formed and constrained within the heteroduplex region of the D-loop. Moreover, the hMSH2-hMSH6 sliding clamps are stabilized on the D-loop by HsRPA bound to the displaced ssDNA. Our findings reveal similarities and differences in hMSH2-hMSH6 mismatch recognition and sliding-clamp formation between a D-loop recombination intermediate and linear duplex DNA.

PSD-95/Dlg/ZO-1 (PDZ) domains are protein-protein interaction modules that generally recognize the C-terminus of target proteins. Here, we investigated the biophysical origin for the novel ligand specificity of a Tiam1 PDZ mutant (QM PDZ) domain that harbors a substitution at four non-conserved residues in the ligand-binding site. Crystal structures of the Tiam1 QM PDZ domain free and bound with two peptide ligands revealed that the four mutations combine to remodel the ligand-binding pocket allowing larger side chains to be accommodated. Importantly, aromatic pi-pi and pi-anion interactions play a central role in ligand recognition. Amide backbone (15N) and methyl side-chain (13C) NMR-based relaxation experiments identified extensive dynamics changes in the QM PDZ domain on both fast (ps-ns) and slow (us-ms) timescales compared to the wild-type PDZ domain. Changes in conformational entropy derived from the analyses of NMR dynamics correlated with changes in binding entropy determined by isothermal titration calorimetry, suggesting that protein dynamics plays a significant role in defining the ligand specificity of the QM PDZ domain. Additionally, biochemical denaturation studies indicated that Tiam1 QM PDZ domain was less thermodynamically stable than the wild-type PDZ domain. Together, our results indicate that structural alterations, increased flexibility and lowered stability combine to support the novel binding specificity of the QM PDZ domain. More broadly, our results support a critical role for conformational dynamics in the evolution of binding specificity.

PDZ (PSD95/Discs large/ZO-1) domains are ubiquitous protein interaction motifs found in scaffolding proteins involved in signal transduction. Despite the fact that many PDZ domains show a limited tendency to undergo structural change, the PDZ family has been associated with long-range communication and allostery. One of the PDZ domains studied most in terms of structure and biophysical properties is the second PDZ ("PDZ2") domain from protein tyrosine phosphatase 1E (PTP1E, also known as PTPL1). Previously, we showed through NMR relaxation studies that binding of the RA-GEF2 C-terminal peptide substrate results in long-range propagation of side-chain dynamic changes in human PDZ2 [Fuentes, E. J., et al. (2004) J. Mol. Biol. 335, 1105-1115]. Here, we present the first X-ray crystal structures of PDZ2 in the absence and presence of RA-GEF2 ligand, determined to resolutions of 1.65 and 1.3 A, respectively. These structures deviate somewhat from previously determined NMR structures and indicate that very minor structural changes in PDZ2 accompany peptide binding. NMR residual dipolar couplings confirm the crystal structures to be accurate models of the time-averaged atomic coordinates of PDZ2. The impact on side-chain dynamics was further tested with a C-terminal peptide from APC, which showed results nearly identical to those of RA-GEF2. Thus, allosteric transmission in PDZ2 induced by peptide binding is conveyed purely and robustly by dynamics. (15)N relaxation dispersion measurements did not detect appreciable populations of a kinetic structural intermediate. Collectively, for ligand binding to PDZ2, these data support a lock-and-key binding model from a structural perspective and an allosteric model from a dynamical perspective, which together suggest a complex energy landscape for functional transitions within the ensemble.