Cryogenic electron microscopy and tomography reveal imperfect icosahedral symmetry in alphaviruses.

Alphaviruses are spherical, enveloped RNA viruses with two-layered icosahedral architecture. The structures of many alphaviruses have been studied using cryogenic electron microscopy (cryo-EM) reconstructions, which impose icosahedral symmetry on the viral particles. Using cryogenic electron tomography (cryo-ET), we revealed a polarized symmetry defect in the icosahedral lattice of Chikungunya virus (CHIKV) in situ, similar to the late budding particles, suggesting the inherent imperfect symmetry originates from the final pinch-off of assembled virions. We further demonstrated this imperfect symmetry is also present in in vitro purified CHIKV and Mayaro virus, another arthritogenic alphavirus. We employed a subparticle-based single-particle analysis protocol to circumvent the icosahedral imperfection and boosted the resolution of the structure of the CHIKV to ∼3 Šresolution, which revealed detailed molecular interactions between glycoprotein E1-E2 heterodimers in the transmembrane region and multiple lipid-like pocket factors located in a highly conserved hydrophobic pocket. This complementary use of in situ cryo-ET and single-particle cryo-EM approaches provides a more precise structural description of near-icosahedral viruses and valuable insights to guide the development of structure-based antiviral therapies against alphaviruses.

Flap endonuclease Rad27 cleaves the RNA of R-loop structures to suppress telomere recombination.

The long non-coding telomeric RNA transcript TERRA, in the form of an RNA-DNA duplex, regulates telomere recombination. In a screen for nucleases that affects telomere recombination, mutations in DNA2, EXO1, MRE11 and SAE2 cause severe delay in type II survivor formation, indicating that type II telomere recombination is mediated through a mechanism similar to repairing double-strand breaks. On the other hand, mutation in RAD27 results in early formation of type II recombination, suggesting that RAD27 acts as a negative regulator in telomere recombination. RAD27 encodes a flap endonuclease that plays a role in DNA metabolism, including replication, repair and recombination. We demonstrate that Rad27 suppresses the accumulation of the TERRA-associated R-loop and selectively cleaves TERRA of R-loop and double-flapped structures in vitro. Moreover, we show that Rad27 negatively regulates single-stranded C-rich telomeric DNA circles (C-circles) in telomerase-deficient cells, revealing a close correlation between R-loop and C-circles during telomere recombination. These results demonstrate that Rad27 participates in telomere recombination by cleaving TERRA in the context of an R-loop or flapped RNA-DNA duplex, providing mechanistic insight into how Rad27 maintains chromosome stability by restricting the accumulation of the R-loop structure within the genome.

Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures.

Both high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open "triplet gate" for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

Promotion of homology-directed DNA repair by polyamines.

Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.

Swi5-Sfr1 stimulates Rad51 recombinase filament assembly by modulating Rad51 dissociation.

Eukaryotic Rad51 protein is essential for homologous-recombination repair of DNA double-strand breaks. Rad51 recombinases first assemble onto single-stranded DNA to form a nucleoprotein filament, required for function in homology pairing and strand exchange. This filament assembly is the first regulation step in homologous recombination. Rad51 nucleation is kinetically slow, and several accessory factors have been identified to regulate this step. Swi5-Sfr1 (S5S1) stimulates Rad51-mediated homologous recombination by stabilizing Rad51 nucleoprotein filaments, but the mechanism of stabilization is unclear. We used single-molecule tethered particle motion experiments to show that mouse S5S1 (mS5S1) efficiently stimulates mouse RAD51 (mRAD51) nucleus formation and inhibits mRAD51 dissociation from filaments. We also used single-molecule fluorescence resonance energy transfer experiments to show that mS5S1 promotes stable nucleus formation by specifically preventing mRAD51 dissociation. This leads to a reduction of nucleation size from three mRAD51 to two mRAD51 molecules in the presence of mS5S1. Compared with mRAD51, fission yeast Rad51 (SpRad51) exhibits fast nucleation but quickly dissociates from the filament. SpS5S1 specifically reduces SpRad51 disassembly to maintain a stable filament. These results clearly demonstrate the conserved function of S5S1 by primarily stabilizing Rad51 on DNA, allowing both the formation of the stable nucleus and the maintenance of filament length.

Role of the RAD51-SWI5-SFR1 Ensemble in homologous recombination.

During DNA double-strand break and replication fork repair by homologous recombination, the RAD51 recombinase catalyzes the DNA strand exchange reaction via a helical polymer assembled on single-stranded DNA, termed the presynaptic filament. Our published work has demonstrated a dual function of the SWI5-SFR1 complex in RAD51-mediated DNA strand exchange, namely, by stabilizing the presynaptic filament and maintaining the catalytically active ATP-bound state of the filament via enhancement of ADP release. In this study, we have strived to determine the basis for physical and functional interactions between Mus musculus SWI5-SFR1 and RAD51. We found that SWI5-SFR1 preferentially associates with the oligomeric form of RAD51. Specifically, a C-terminal domain within SWI5 contributes to RAD51 interaction. With specific RAD51 interaction defective mutants of SWI5-SFR1 that we have isolated, we show that the physical interaction is indispensable for the stimulation of the recombinase activity of RAD51. Our results thus help establish the functional relevance of the trimeric RAD51-SWI5-SFR1 complex and provide insights into the mechanistic underpinnings of homology-directed DNA repair in mammalian cells.

Functional Relationship of ATP Hydrolysis, Presynaptic Filament Stability, and Homologous DNA Pairing Activity of the Human Meiotic Recombinase DMC1.

DMC1 and RAD51 are conserved recombinases that catalyze homologous recombination. DMC1 and RAD51 share similar properties in DNA binding, DNA-stimulated ATP hydrolysis, and catalysis of homologous DNA strand exchange. A large body of evidence indicates that attenuation of ATP hydrolysis leads to stabilization of the RAD51-ssDNA presynaptic filament and enhancement of DNA strand exchange. However, the functional relationship of ATPase activity, presynaptic filament stability, and DMC1-mediated homologous DNA strand exchange has remained largely unexplored. To address this important question, we have constructed several mutant variants of human DMC1 and characterized them biochemically to gain mechanistic insights. Two mutations, K132R and D223N, that change key residues in the Walker A and B nucleotide-binding motifs ablate ATP binding and render DMC1 inactive. On the other hand, the nucleotide-binding cap D317K mutant binds ATP normally but shows significantly attenuated ATPase activity and, accordingly, forms a highly stable presynaptic filament. Surprisingly, unlike RAD51, presynaptic filament stabilization achieved via ATP hydrolysis attenuation does not lead to any enhancement of DMC1-catalyzed homologous DNA pairing and strand exchange. This conclusion is further supported by examining wild-type DMC1 with non-hydrolyzable ATP analogues. Thus, our results reveal an important mechanistic difference between RAD51 and DMC1.

Enhancement of ADP release from the RAD51 presynaptic filament by the SWI5-SFR1 complex.

Homologous recombination catalyzed by the RAD51 recombinase eliminates deleterious DNA lesions from the genome. In the presence of ATP, RAD51 forms a nucleoprotein filament on single-stranded DNA, termed the presynaptic filament, to initiate homologous recombination-mediated DNA double-strand break repair. The SWI5-SFR1 complex stabilizes the presynaptic filament and enhances its ability to mediate the homologous DNA pairing reaction. Here we characterize the RAD51 presynaptic filament stabilization function of the SWI5-SFR1 complex using optical tweezers. Biochemical experiments reveal that SWI5-SFR1 enhances ATP hydrolysis by single-stranded DNA-bound RAD51. Importantly, we show that SWI5-SFR1 acts by facilitating the release of ADP from the presynaptic filament. Our results thus provide mechanistic understanding of the function of SWI5-SFR1 in RAD51-mediated DNA recombination.

Systems biology of meridians, acupoints, and chinese herbs in disease.

Meridians, acupoints, and Chinese herbs are important components of traditional Chinese medicine (TCM). They have been used for disease treatment and prevention and as alternative and complementary therapies. Systems biology integrates omics data, such as transcriptional, proteomic, and metabolomics data, in order to obtain a more global and complete picture of biological activity. To further understand the existence and functions of the three components above, we reviewed relevant research in the systems biology literature and found many recent studies that indicate the value of acupuncture and Chinese herbs. Acupuncture is useful in pain moderation and relieves various symptoms arising from acute spinal cord injury and acute ischemic stroke. Moreover, Chinese herbal extracts have been linked to wound repair, the alleviation of postmenopausal osteoporosis severity, and anti-tumor effects, among others. Different acupoints, variations in treatment duration, and herbal extracts can be used to alleviate various symptoms and conditions and to regulate biological pathways by altering gene and protein expression. Our paper demonstrates how systems biology has helped to establish a platform for investigating the efficacy of TCM in treating different diseases and improving treatment strategies.

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex.

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.

NADH fluorescence as a photobiological metric in 5-aminolevlinic acid (ALA)-photodynamic therapy.

Photodynamic therapy (PDT) dosimetry is complex as many factors are involved and varied interdependently. Monitoring the biological consequence of PDT such as cell death is the most direct approach to assess treatment efficacy. In this study, we performed 5-aminolevlinic acid (ALA)-PDT in vitro to induce different cell death modes (i.e., slight cell cytotoxicity, apoptosis, and necrosis) by a fixed fluence rate of 10 mW/cm(2) and varied fluences (1, 2, and 6 J/cm(2)). Time course measurements of cell viability, caspase-3 activity, and DNA fragmentation were conducted to determine the mode of cell death. We demonstrated that NADH fluorescence lifetime together with NADH fluorescence intensity permit us to detect apoptosis and differentiate it from necrosis. This feature will be unique in the use of optimizing apoptosis-favored treatments such as metronomic PDT.