Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

A Reversible Association between Smc Coiled Coils Is Regulated by Lysine Acetylation and Is Required for Cohesin Association with the DNA.

Cohesin is a ring-shaped protein complex that is capable of embracing DNA. Most of the ring circumference is comprised of the anti-parallel intramolecular coiled coils of the Smc1 and Smc3 proteins, which connect globular head and hinge domains. Smc coiled coil arms contain multiple acetylated and ubiquitylated lysines. To investigate the role of these modifications, we substituted lysines for arginines to mimic the unmodified state and uncovered genetic interaction between the Smc arms. Using scanning force microscopy, we show that wild-type Smc arms associate with each other when the complex is not on DNA. Deacetylation of the Smc1/Smc3 dimers promotes arms' dissociation. Smc arginine mutants display loose packing of the Smc arms and, although they dimerize at the hinges, fail to connect the heads and associate with the DNA. Our findings highlight the importance of a "collapsed ring," or "rod," conformation of cohesin for its loading on the chromosomes.

Experimental systems for the analysis of mutational signatures: no 'one-size-fits-all' solution.

Cells constantly accumulate mutations, which are caused by replication errors, as well as through the action of endogenous and exogenous DNA-damaging agents. Mutational patterns reflect the status of DNA repair machinery and the history of genotoxin exposure of a given cellular clone. Computationally derived mutational signatures can shed light on the origins of cancer. However, to understand the etiology of cancer signatures, they need to be compared with experimental signatures, which are obtained from the isogenic cell lines or organisms under controlled conditions. Experimental mutational patterns were instrumental in understanding the nature of signatures caused by mismatch repair and BRCA deficiencies. Here, we describe how different cell lines and model organisms were used in recent years to decipher mutational signatures observed in cancer genomes and provide examples of how data from different experimental systems complement and support each other.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

Structural Insight into the DNA Binding Function of Transcription Factor ERF.

ETS family transcription factors control development of different cell types in humans, whereas deregulation of these proteins leads to severe developmental syndromes and cancers. One of a few members of the ETS family that are known to act solely as repressors, ERF, is required for normal osteogenesis and hematopoiesis. Another important function of ERF is acting as a tumor suppressor by antagonizing oncogenic fusions involving other ETS family factors. The structure of ERF and the DNA binding properties specific to this protein have not been elucidated. In this study, we determined two crystal structures of the complexes of the DNA binding domain of ERF with DNA. In one, ERF is in a distinct dimeric form, with Cys72 in a reduced state. In the other, two dimers of ERF are assembled into a tetramer that is additionally locked by two Cys72-Cys72 disulfide bonds across the dimers. In the tetramer, the ERF molecules are bound to a pseudocontinuous DNA on the same DNA face at two GGAA binding sites on opposite strands. Sedimentation velocity analysis showed that this tetrameric assembly forms on continuous DNA containing such tandem sites spaced by 7 bp. Our bioinformatic analysis of three previously reported sets of ERF binding loci across entire genomes showed that these loci were enriched in such 7 bp spaced tandem sites. Taken together, these results strongly suggest that the observed tetrameric assembly is a functional state of ERF in the human cell.

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis.

MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer-Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso-mithramycin (iso-MTM). Iso-MTM undergoes a non-enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co-substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring-shaped ß subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso-MTM, as a potential self-resistance mechanism against MTM toxicity.

General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity (KD of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity (KD of ∼10 µM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition.IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly through its cyclophilin A domain and therefore was thought to act independently of higher-order assembly. Here, we show that TRIMCyp shares the assembly properties of TRIM5α and that both forms of TRIM5 use the same mechanism of hexagonal lattice formation to promote viral recognition and restriction.

Universal many-body response of heavy impurities coupled to a Fermi sea: a review of recent progress.

In this report we discuss the dynamical response of heavy quantum impurities immersed in a Fermi gas at zero and at finite temperature. Studying both the frequency and the time domain allows one to identify interaction regimes that are characterized by distinct many-body dynamics. From this theoretical study a picture emerges in which impurity dynamics is universal on essentially all time scales, and where the high-frequency few-body response is related to the long-time dynamics of the Anderson orthogonality catastrophe by Tan relations. Our theoretical description relies on different and complementary approaches: functional determinants give an exact numerical solution for time- and frequency-resolved responses, bosonization provides accurate analytical expressions at low temperatures, and the theory of Toeplitz determinants allows one to analytically predict response up to high temperatures. Using these approaches we predict the thermal decoherence rate of the fermionic system and prove that within the considered model the fastest rate of long-time decoherence is given by [Formula: see text]. We show that Feshbach resonances in cold atomic systems give access to new interaction regimes where quantum effects can prevail even in the thermal regime of many-body dynamics. The key signature of this phenomenon is a crossover between different exponential decay rates of the real-time Ramsey signal. It is shown that the physics of the orthogonality catastrophe is experimentally observable up to temperatures [Formula: see text] where it leaves its fingerprint in a power-law temperature dependence of thermal spectral weight and we review how this phenomenon is related to the physics of heavy ions in liquid [Formula: see text]He and the formation of Fermi polarons. The presented results are in excellent agreement with recent experiments on LiK mixtures, and we predict several new phenomena that can be tested using currently available experimental technology.

Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells.

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.

Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus.

IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell-dependent and T cell-independent Ab responses, but it did not affect T cell-mediated clearance of Chlamydia infection, consistent with a B cell-specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.

Structures of mithramycin analogues bound to DNA and implications for targeting transcription factor FLI1.

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

A SAMHD1 mutation associated with Aicardi-Goutières syndrome uncouples the ability of SAMHD1 to restrict HIV-1 from its ability to downmodulate type I interferon in humans.

Mutations in the human SAMHD1 gene are known to correlate with the development of the Aicardi-Goutières syndrome (AGS), which is an inflammatory encephalopathy that exhibits neurological dysfunction characterized by increased production of type I interferon (IFN); this evidence has led to the concept that the SAMHD1 protein negatively regulates the type I IFN response. Additionally, the SAMHD1 protein has been shown to prevent efficient HIV-1 infection of macrophages, dendritic cells, and resting CD4+ T cells. To gain insights on the SAMHD1 molecular determinants that are responsible for the deregulated production of type I IFN, we explored the biochemical, cellular, and antiviral properties of human SAMHD1 mutants known to correlate with the development of AGS. Most of the studied SAMHD1 AGS mutants exhibit defects in the ability to oligomerize, decrease the levels of cellular deoxynucleotide triphosphates in human cells, localize exclusively to the nucleus, and restrict HIV-1 infection. At least half of the tested variants preserved the ability to be degraded by the lentiviral protein Vpx, and all of them interacted with RNA. Our investigations revealed that the SAMHD1 AGS variant p.G209S preserve all tested biochemical, cellular, and antiviral properties, suggesting that this residue is a determinant for the ability of SAMHD1 to negatively regulate the type I IFN response in human patients with AGS. Overall, our work genetically separated the ability of SAMHD1 to negatively regulate the type I IFN response from its ability to restrict HIV-1.

Allosteric Activation of SAMHD1 Protein by Deoxynucleotide Triphosphate (dNTP)-dependent Tetramerization Requires dNTP Concentrations That Are Similar to dNTP Concentrations Observed in Cycling T Cells.

SAMHD1 is a dNTP hydrolase, whose activity is required for maintaining low dNTP concentrations in non-cycling T cells, dendritic cells, and macrophages. SAMHD1-dependent dNTP depletion is thought to impair retroviral replication in these cells, but the relationship between the dNTPase activity and retroviral restriction is not fully understood. In this study, we investigate allosteric activation of SAMHD1 by deoxynucleotide-dependent tetramerization and measure how the lifetime of the enzymatically active tetramer is affected by different dNTP ligands bound in the allosteric site. The EC50dNTP values for SAMHD1 activation by dNTPs are in the 2-20 µm range, and the half-life of the assembled tetramer after deoxynucleotide depletion varies from minutes to hours depending on what dNTP is bound in the A2 allosteric site. Comparison of the wild-type SAMHD1 and the T592D mutant reveals that the phosphomimetic mutation affects the rates of tetramer dissociation, but has no effect on the equilibrium of allosteric activation by deoxynucleotides. Collectively, our data suggest that deoxynucleotide-dependent tetramerization contributes to regulation of deoxynucleotide levels in cycling cells, whereas in non-cycling cells restrictive to retroviral replication, SAMHD1 activation is likely to be achieved through a distinct mechanism.

A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability.

SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592 Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells.

Structural basis of HIV-1 capsid recognition by PF74 and CPSF6.

Upon infection of susceptible cells by HIV-1, the conical capsid formed by ∼250 hexamers and 12 pentamers of the CA protein is delivered to the cytoplasm. The capsid shields the RNA genome and proteins required for reverse transcription. In addition, the surface of the capsid mediates numerous host-virus interactions, which either promote infection or enable viral restriction by innate immune responses. In the intact capsid, there is an intermolecular interface between the N-terminal domain (NTD) of one subunit and the C-terminal domain (CTD) of the adjacent subunit within the same hexameric ring. The NTD-CTD interface is critical for capsid assembly, both as an architectural element of the CA hexamer and pentamer and as a mechanistic element for generating lattice curvature. Here we report biochemical experiments showing that PF-3450074 (PF74), a drug that inhibits HIV-1 infection, as well as host proteins cleavage and polyadenylation specific factor 6 (CPSF6) and nucleoporin 153 kDa (NUP153), bind to the CA hexamer with at least 10-fold higher affinities compared with nonassembled CA or isolated CA domains. The crystal structure of PF74 in complex with the CA hexamer reveals that PF74 binds in a preformed pocket encompassing the NTD-CTD interface, suggesting that the principal inhibitory target of PF74 is the assembled capsid. Likewise, CPSF6 binds in the same pocket. Given that the NTD-CTD interface is a specific molecular signature of assembled hexamers in the capsid, binding of NUP153 at this site suggests that key features of capsid architecture remain intact upon delivery of the preintegration complex to the nucleus.

Histone H3 localizes to the centromeric DNA in budding yeast.

During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.

Cohesin rings devoid of Scc3 and Pds5 maintain their stable association with the DNA.

Cohesin is a protein complex that forms a ring around sister chromatids thus holding them together. The ring is composed of three proteins: Smc1, Smc3 and Scc1. The roles of three additional proteins that associate with the ring, Scc3, Pds5 and Wpl1, are not well understood. It has been proposed that these three factors form a complex that stabilizes the ring and prevents it from opening. This activity promotes sister chromatid cohesion but at the same time poses an obstacle for the initial entrapment of sister DNAs. This hindrance to cohesion establishment is overcome during DNA replication via acetylation of the Smc3 subunit by the Eco1 acetyltransferase. However, the full mechanistic consequences of Smc3 acetylation remain unknown. In the current work, we test the requirement of Scc3 and Pds5 for the stable association of cohesin with DNA. We investigated the consequences of Scc3 and Pds5 depletion in vivo using degron tagging in budding yeast. The previously described DHFR-based N-terminal degron as well as a novel Eco1-derived C-terminal degron were employed in our study. Scc3 and Pds5 associate with cohesin complexes independently of each other and require the Scc1 "core" subunit for their association with chromosomes. Contrary to previous data for Scc1 downregulation, depletion of either Scc3 or Pds5 had a strong effect on sister chromatid cohesion but not on cohesin binding to DNA. Quantity, stability and genome-wide distribution of cohesin complexes remained mostly unchanged after the depletion of Scc3 and Pds5. Our findings are inconsistent with a previously proposed model that Scc3 and Pds5 are cohesin maintenance factors required for cohesin ring stability or for maintaining its association with DNA. We propose that Scc3 and Pds5 specifically function during cohesion establishment in S phase.

Structure of the rhesus monkey TRIM5α PRYSPRY domain, the HIV capsid recognition module.

Tripartite motif protein TRIM5α blocks retroviral replication after cell entry, and species-specific differences in its activity are determined by sequence variations within the C-terminal B30.2/PRYSPRY domain. Here we report a high-resolution structure of a TRIM5α PRYSPRY domain, the PRYSPRY of the rhesus monkey TRIM5α that potently restricts HIV infection, and identify features involved in its interaction with the HIV capsid. The extensive capsid-binding interface maps on the structurally divergent face of the protein formed by hypervariable loop segments, confirming that TRIM5α evolution is largely determined by its binding specificity. Interactions with the capsid are mediated by flexible variable loops via a mechanism that parallels antigen recognition by IgM antibodies, a similarity that may help explain some of the unusual functional properties of TRIM5α. Distinctive features of this pathogen-recognition interface, such as structural plasticity conferred by the mobile v1 segment and interaction with multiple epitopes, may allow restriction of divergent retroviruses and increase resistance to capsid mutations.

Recognition of the HIV capsid by the TRIM5α restriction factor is mediated by a subset of pre-existing conformations of the TRIM5α SPRY domain.

The binding of the TRIM5α restriction factor to the HIV capsid is mediated by the C-terminal SPRY domain of TRIM5α. Atomic-level details of this host-pathogen interaction, which involves mobile variable loops of the SPRY domain, remain unclear. Some of the key determinants of restriction are encompassed by the long and disordered v1 loop of the SPRY domain. We applied molecular modeling to elucidate the conformational repertoire of the v1 loop and its role in the interaction with the capsid. All-atom replica exchange molecular dynamics revealed multiple transient, interconverting states of the v1 loop consistent with the intrinsic disorder observed experimentally. The docking of the SPRY conformations representing 10 most populated states onto the high-resolution model of the assembled HIV-1 capsid revealed that a subset of v1 conformations produced plausible binding poses, in which the SPRY domain binds close to the pseudo-2-fold symmetry axis and the v1 loop spans the interhexamer gap. Such binding mode is well supported by the NMR binding data and known escape mutants. We speculate that the binding mode that involves interaction of the capsid with a subset of preexisting SPRY conformations arising from the intrinsic disorder of the v1 loop may explain the remarkable ability of TRIM5α to resist viral evasion by mutagenesis and to restrict divergent retroviruses.

Trabectedin derails transcription-coupled nucleotide excision repair to induce DNA breaks in highly transcribed genes.

Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription.