A pentameric protein ring with novel architecture is required for herpesviral packaging

Genome packaging in large double-stranded DNA viruses requires a powerful molecular motor to force the viral genome into nascent capsids, which involves essential accessory factors that are poorly understood. Here, we present structures of two such accessory factors from the oncogenic herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV; ORF68) and Epstein–Barr virus (EBV; BFLF1). These homologous proteins form highly similar homopentameric rings with a positively charged central channel that binds double-stranded DNA. Mutation of individual positively charged residues within but not outside the channel ablates DNA binding, and in the context of KSHV infection, these mutants fail to package the viral genome or produce progeny virions. Thus, we propose a model in which ORF68 facilitates the transfer of newly replicated viral genomes to the packaging motor.

Believing that transferring more embryos will result in increased pregnancy rates: a flawed concept: a SWOT analysis

A belief exists that transferring multiple embryos can improve pregnancy rates. However, this concept is flawed. Multiple factors affect outcomes when transferring embryos, endometrial receptivity, and rates of aneuploidy among them. In this article, we will discuss how factors can affect outcomes at IVF that are independent of the number of embryos transferred. It is important to understand the role of accessory factors on pregnancy rates to be able to counsel patients as per the number of embryos that should be transferred. An understanding of this concept will also lead to a realistic understanding of how multiple embryo transfers may result in better cumulative pregnancy rates than a single transfer of multiple embryos. Finally, we will present a SWOT analysis diagram to help guide clinical decision-making.

Retromer forms low order oligomers on supported lipid bilayers

Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)–retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (∼18%), dimer (∼35%), trimer (∼24%), and tetramer (∼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.

Chromatin Architectural Factors as Safeguards against Excessive Supercoiling during DNA Replication

Key DNA transactions, such as genome replication and transcription, rely on the speedy translocation of specialized protein complexes along a double-stranded, right-handed helical template. Physical tethering of these molecular machines during translocation, in conjunction with their internal architectural features, generates DNA topological strain in the form of template supercoiling. It is known that the build-up of transient excessive supercoiling poses severe threats to genome function and stability and that highly specialized enzymes—the topoisomerases (TOP)—have evolved to mitigate these threats. Furthermore, due to their intracellular abundance and fast supercoil relaxation rates, it is generally assumed that these enzymes are sufficient in coping with genome-wide bursts of excessive supercoiling. However, the recent discoveries of chromatin architectural factors that play important accessory functions have cast reasonable doubts on this concept. Here, we reviewed the background of these new findings and described emerging models of how these accessory factors contribute to supercoil homeostasis. We focused on DNA replication and the generation of positive (+) supercoiling in front of replisomes, where two accessory factors—GapR and HMGA2—from pro- and eukaryotic cells, respectively, appear to play important roles as sinks for excessive (+) supercoiling by employing a combination of supercoil constrainment and activation of topoisomerases. Looking forward, we expect that additional factors will be identified in the future as part of an expanding cellular repertoire to cope with bursts of topological strain. Furthermore, identifying antagonists that target these accessory factors and work synergistically with clinically relevant topoisomerase inhibitors could become an interesting novel strategy, leading to improved treatment outcomes.

Retromer forms low order oligomers on supported lipid bilayers

Retromer is a protein sorting device that orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long standing hypotheses regarding the Retromer sorting mechanism posit that oligomeric interactions between Retromer and associated accessory factors on the endosome membrane drives clustering of Retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this hypothesis, we examined interactions between the components of the SNX3-Retromer sorting pathway using quantitative single particle fluorescence microscopy of a reconstituted system comprising a supported bilayer, Retromer, a model cargo protein, the accessory proteins SNX3, RAB7, and the Retromer-binding segment of the WASHC2C subunit of the WASH complex. The predominant species of membrane associated Retromer are low order: monomers (∼18%), dimers (∼35%), trimers (∼24%) and tetramers (∼24%). Unexpectedly, neither cargo nor accessory factors promote Retromer oligomerization on a supported bilayer. The results indicate that Retromer has an intrinsic propensity to form low order oligomers and that neither membrane association nor accessory factors potentiate oligomerization. Hence, Retromer is a minimally concentrative sorting device adapted to bulk membrane trafficking from the endosomal system.

Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes, and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. The Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factor subunits X-ray repair cross-complementing group 4 (XRCC4), XRCC4-like factor (XLF), and DNA ligase 4 (Lig4). Accessory factors might be dispensable for the process, depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced a frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as the wild type controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

Classical non-homologous end joining (NHEJ) is a molecular pathway that detects, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. Mutations in several NHEJ genes result in neurological abnormalities and immunodeficiency both in humans and mice. The NHEJ pathway is required for the V(D)J recombination in developing B and T lymphocytes, and for class switch recombination in mature B cells. Ku heterodimer formed by Ku70 and Ku80 recognizes DSBs and facilitates the recruitment of accessory factors (e.g., DNA-PKcs, Artemis, Paxx and Mri/Cyren) and downstream core factors subunits XLF, XRCC4 and Lig4. Accessory factors might be dispensable for the process depending on the genetic background and DNA lesion type. To determine the physiological role of Mri in DNA repair and development, we introduced frame-shift mutation in the Mri gene in mice. We then analyzed the development of Mri-deficient mice as well as wild type and immunodeficient controls. Mice lacking Mri possessed reduced levels of class switch recombination in B lymphocytes and slow proliferation of neuronal progenitors when compared to wild type littermates. Human cell lines lacking Mri were as sensitive to DSBs as WT controls. Overall, we concluded that Mri/Cyren is largely dispensable for DNA repair and mouse development.

A mutant form of Dmc1 that bypasses the requirement for accessory protein Mei5-Sae3 reveals independent activities of Mei5-Sae3 and Rad51 in Dmc1 filament stability

During meiosis, homologous recombination repairs programmed DNA double-stranded breaks (DSBs). Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. We also provide evidence that Mei5-Sae3 promotes Dmc1 filament formation specifically on single-stranded DNA. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister (IS) joint molecules intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1 (Dmc1-WT). Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filaments lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.Author SummaryDuring meiosis, two rounds of division follow a single round of DNA replication to create the gametes for biparental reproduction. The first round of division requires that the homologous chromosomes become physically linked to one another to create the tension that is necessary for their segregation. This linkage is achieved through DNA recombination between the two homologous chromosomes, followed by resolution of the recombination intermediate into a crossover (CO). Central to this process is the meiosis-specific recombinase Dmc1, and its accessory factors, which provide important regulatory functions to ensure that recombination is accurate, efficient, and occurs predominantly between homologous chromosomes, and not sister chromatids. To gain insight into the regulation of Dmc1 by its accessory factors, we mutated Dmc1 such that it was no longer dependent on its accessory factor Mei5-Sae3. Our analysis reveals that Dmc1 accessory factors Mei5-Sae3 and Rad51 have independent roles in stabilizing Dmc1 filaments. Furthermore, we find that although Rad51 is required for promoting recombination between homologous chromosomes, Mei5-Sae3 is not. Lastly, we show that our Dmc1 mutant forms abnormally long filaments, and high levels of aberrant recombination intermediates and products. These findings suggest that filaments are actively maintained at short lengths to prevent deleterious genome rearrangements.

Intrinsic and regulated properties of minimally edited trypanosome mRNAs

Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing. We show that CYb and MURF2 mRNAs exhibit barriers to editing progression that differ from those previously identified for pan-edited mRNAs, primarily at initial gRNA usage and gRNA exchange. We demonstrate that mis-edited junctions arise through multiple pathways including mis-alignment of cognate gRNA, incorrect and sometimes promiscuous gRNA utilization and inefficient gRNA anchoring. We then examined the roles of accessory factors RBP16 and MRP1/2 in maintaining edited CYb and MURF2 populations. RBP16 is essential for initiation of CYb and MURF2 editing, as well as MURF2 editing progression. In contrast, MRP1/2 stabilizes both edited mRNA populations, while further promoting progression of MURF2 mRNA editing. We also analyzed the effects of RNA Editing Substrate Binding Complex components, TbRGG2 and GAP1, and show that both proteins modestly impact progression of editing on minimally edited mRNAs, suggesting a novel function for GAP1.