Single-Molecule Studies of Origin Licensing Reveal Mechanisms Ensuring Bidirectional Helicase Loading

Loading of the ring-shaped Mcm2–7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2–7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2–7 complexes. Loading of each Mcm2–7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2–7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation.National Institutes of Health (U.S.) (NIH grant GM52339)National Institutes of Health (U.S.) (NIH grant R01 GM81648)G. Harold and Leila Y. Mathers FoundationNational Institutes of Health (U.S.) (NIH Pre-Doctoral Training Grant (GM007287))Howard Hughes Medical Institute (Investigator

Mechanism and timing of Mcm2–7 ring closure during DNA replication origin licensing

The opening and closing of two ring-shaped Mcm2-7 DNA helicases is necessary to license eukaryotic origins of replication, although the mechanisms controlling these events are unclear. The origin-recognition complex (ORC), Cdc6 and Cdt1 facilitate this process by establishing a topological link between each Mcm2-7 hexamer and origin DNA. Using colocalization single-molecule spectroscopy and single-molecule Förster resonance energy transfer (FRET), we monitored ring opening and closing of Saccharomyces cerevisiae Mcm2-7 during origin licensing. The two Mcm2-7 rings were open during initial DNA association and closed sequentially, concomitant with the release of their associated Cdt1. We observed that ATP hydrolysis by Mcm2-7 was coupled to ring closure and Cdt1 release, and failure to load the first Mcm2-7 prevented recruitment of the second Mcm2-7. Our findings identify key mechanisms controlling the Mcm2-7 DNA-entry gate during origin licensing, and reveal that the two Mcm2-7 complexes are loaded via a coordinated series of events with implications for bidirectional replication initiation and quality control.National Institutes of Health (U.S.) (Grant R01 GM52339)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

Single-molecule studies of eukaryotic helicase loading

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Cells must duplicate their genomic content fully and accurately in each cell cycle to maintain cellular identity and ensure the viability of their progeny. The first step in eukaryotic DNA replication initiation is the loading of the heterohexameric Mcm2-7 helicase. In eukaryotes helicase loading is tightly regulated to ensure that this event only occurs during the GI phase of the cell cycle. Because helicase activation can only occur once cells enter S phase, cells can only load and activate the helicase once per cell cycle, ensuring the genome is replicated once and only once in each cell cycle. The selection of sites of helicase-loading also marks all potential origins of replication. Once loaded, the helicases encircle dsDNA and are linked in a head-to-head double hexamer. Although the proteins involved in helicase loading are known (ORC, Cdc6, and Cdt 1), the mechanism by which they load two oppositely-oriented helicases and ensure their proper architecture remains under intense investigation. In this thesis I describe a novel single-molecule helicase-loading assay that allows monitoring of protein associations and dissociations on a one-second time scale. By labeling pairs of helicase-loading proteins simultaneously, I determined the relative time of association and dissociation for the helicase and each of the helicase-loading proteins during helicase loading. Additionally, I determined the stoichiometry of each helicase-loading protein with respect to the origin DNA. Adapting this assay to read out distance information using single-molecule FRET, I monitored formation of the final double-hexamer in real time. These single-molecule assays uncovered that helicase loading occurs in a one-at-a- time manner and discovered novel steps in the mechanism of helicase loading. Following the initial association of ORC/Cdc6/Cdtl/Mcm2-7 with the origins of DNA replication, Cdc6 and then Cdtl are released sequentially. A new Cdc6, and Cdtl/Mcm2- 7 are subsequently recruited and the same ordered sequential release is observed, although with different kinetics. Although two Cdc6 and Cdtl proteins are required for loading a double hexamer, a single ORC is sufficient. Additionally, double-hexamer formation is a rapid event upon association of the second helicase, suggesting a model in which the two helicases are recruited and loaded around dsDNA by distinct mechanisms.by Simina Ticau.Ph. D

Baculovirus AC102 Is a Nucleocapsid Protein That Is Crucial for Nuclear Actin Polymerization and Nucleocapsid Morphogenesis

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type species of alphabaculoviruses, is an enveloped DNA virus that infects lepidopteran insects and is commonly known as a vector for protein expression and cell transduction. AcMNPV belongs to a diverse group of viral and bacterial pathogens that target the host cell actin cytoskeleton during infection. AcMNPV is unusual, however, in that it absolutely requires actin translocation into the nucleus early in infection and actin polymerization within the nucleus late in infection coincident with viral replication. Of the six viral factors that are sufficient, when coexpressed, to induce the nuclear localization of actin, only AC102 is essential for viral replication and the nuclear accumulation of actin. We therefore sought to better understand the role of AC102 in actin mobilization in the nucleus early and late in infection. Although AC102 was proposed to function early in infection, we found that AC102 is predominantly expressed as a late protein. In addition, we observed that AC102 is required for F-actin assembly in the nucleus during late infection, as well as for proper formation of viral replication structures and nucleocapsid morphogenesis. Finally, we found that AC102 is a nucleocapsid protein and a newly recognized member of a complex consisting of the viral proteins EC27, C42, and the actin polymerization protein P78/83. Taken together, our findings suggest that AC102 is necessary for nucleocapsid morphogenesis and actin assembly during late infection through its role as a component of the P78/83-C42-EC27-AC102 protein complex.IMPORTANCE The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an important biotechnological tool for protein expression and cell transduction, and related nucleopolyhedroviruses are also used as environmentally benign insecticides. One impact of our work is to better understand the fundamental mechanisms through which AcMNPV exploits the cellular machinery of the host for replication, which may aid in the development of improved baculovirus-based research and industrial tools. Moreover, AcMNPV's ability to mobilize the host actin cytoskeleton within the cell's nucleus during infection makes it a powerful cell biological tool. It is becoming increasingly clear that actin plays important roles in the cell's nucleus, and yet the regulation and function of nuclear actin is poorly understood. Our work to better understand how AcMNPV relocalizes and polymerizes actin within the nucleus may reveal fundamental mechanisms that govern nuclear actin regulation and function, even in the absence of viral infection

The Dynamics of Eukaryotic Replication Initiation: Origin Specificity, Licensing, and Firing at the Single-Molecule Level

Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2–7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2–7 loading specificity and that Mcm2–7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2–7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes.National Institutes of Health (U.S.) (Grant GM52339)National Institutes of Health (U.S.) (Pre-Doctoral Training Grant GM007287)National Science Foundation (U.S.) (Graduate Research Fellowship 1122374

Treatment response and neurofilament light chain levels with long-term patisiran in hereditary transthyretin-mediated amyloidosis with polyneuropathy: 24-month results of an open-label extension study

Longitudinal changes in neurofilament light chain (NfL) levels were evaluated alongside prespecified clinical assessments 24 months into the patisiran Global open-label extension (OLE) study in patients with ATTRv amyloidosis with polyneuropathy. All patients enrolled in the Global OLE, from phase III APOLLO and phase II OLE parent studies, received patisiran. Assessments included measures of polyneuropathy (modified Neuropathy Impairment Score+7 (mNIS+7)), quality of life (QOL; Norfolk QOL-Diabetic Neuropathy questionnaire (Norfolk QOL-DN)), and plasma NfL. Patients receiving patisiran in the parent study (APOLLO-patisiran, n = 137; phase II OLE-patisiran, n = 25) demonstrated sustained improvements in mNIS+7 (mean change from parent study baseline (95% confidence interval): APOLLO-patisiran −4.8 (−8.9, −0.6); phase II OLE-patisiran −5.8 (−10.5, −1.2)) and Norfolk QOL-DN (APOLLO-patisiran −2.4 (−7.2, 2.3)), and maintained reduced NfL levels at Global OLE 24 months. After initiating patisiran in the Global OLE, APOLLO-placebo patients (n = 49) demonstrated stabilized mNIS+7, improved Norfolk QOL-DN, and significantly reduced NfL levels. Patisiran continued to demonstrate an acceptable safety profile. Earlier patisiran initiation was associated with a lower exposure-adjusted mortality rate. Long-term patisiran treatment led to sustained improvements in neuropathy and QOL, with NfL demonstrating potential as a biomarker for disease progression and treatment response in ATTRv amyloidosis with polyneuropathy.</p

Neurofilament light chain (NfL) as a biomarker of hereditary transthyretin-mediated amyloidosis

To identify changes in the proteome associated with onset and progression of ATTRv amyloidosis, we performed an observational, case-controlled study which compared proteomes of patients with ATTRv amyloidosis and healthy controls. Plasma levels of >1,000 proteins were measured in patients with ATTRv amyloidosis with polyneuropathy who received either placebo or patisiran in the APOLLO study and in healthy controls. The impact of patisiran on the time profile of each protein was determined by linear mixed model at 0, 9, and 18 months. Neurofilament light chain (NfL) was further assessed using an orthogonal quantitative approach. Levels of 66 proteins were significantly changed with patisiran vs placebo, with NfL change most significant (p < 10−20). Analysis of changes in protein levels demonstrated that the proteome of patisiran-treated patients trended toward healthy controls at 18 months. Healthy controls' NfL levels were 4-fold lower than in patients with ATTRv amyloidosis with polyneuropathy (16.3 vs 69.4 pg/mL, effect: −53.1 pg/mL, 95% CI [–60.5 to −45.9]). NfL levels at 18 months increased with placebo (99.5 vs 63.2 pg/mL, 36.3 pg/mL, [16.5–56.1]) and decreased with patisiran treatment (48.8 vs 72.1 pg/mL, −23.3 pg/mL, [–33.4 to −13.1]) from baseline. At 18 months, improvement in modified Neuropathy Impairment Score +7 following patisiran significantly correlated with reduced NfL (R = 0.43, [0.29–0.55]). Findings suggest NfL may serve as a biomarker of nerve damage and polyneuropathy in ATTRv amyloidosis, may enable earlier diagnosis of patients with ATTRv amyloidosis, and facilitate monitoring of disease progression. This study provides Class III evidence that NfL levels may enable earlier diagnosis of polyneuropathy in patients with ATTRv amyloidosis and facilitate monitoring of disease progression.This supplementary data comprises: Table e-1 A comprehensive list of all proteins that significantly changed between placebo- and patisiran-treated hereditary transthyretin (ATTRv) amyloidosis patients over time during the APOLLO study Table e-2 Comparisons of levels of neurofilament light chain (NfL) between healthy controls and placebo- or patisiran-treated patients at baseline, 9 months, or 18 months Table e-3 Comparisons of levels of NfL in baseline APOLLO patients at different polyneuropathy disability (PND) scores Table e-4 Comparisons of levels of NfL in baseline APOLLO patients that were in the prespecified cardiac subpopulation or not to healthy controls Table e-5 Comparisons of levels of NfL in healthy controls and placebo- or patisiran-treated patients at baseline or 18 months between groups