Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis
Zachary D. Nagel, Carrie M. Margulies, Isaac A. Chaim, Siobhan K. McRee, Patrizia Mazzucato, Anwaar Ahmad, Ryan P. Abo, Vincent L. Butty, Anthony L. Forget, and Leona D. Samson
DNA, the blueprint of the cell, is constantly damaged by chemicals and radiation. Because DNA damage may cause cell death or mutations that may lead to diseases such as cancer, cells are armed with an arsenal of several distinct mechanisms for repairing the many types of DNA damage that occur. DNA repair capacity (DRC) varies among individuals, and reduced DRC is associated with disease risk; however, the available DRC assays are labor intensive and measure only one pathway at a time. Herein (pp. E1823–E1832), we present powerful new assays that measure DRC in multiple pathways in a single assay. We use the assays to measure interindividual DRC differences and inhibition of DNA repair, and to uncover unexpected error-prone transcriptional bypass of a thymine dimer.
Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load
Yasuharu Takagi, Rachel E. Farrow, Neil Billington, Attila Nagy, Christopher Batters, Yi Yang, James R. Sellers, and Justin E. Molloy
Filopodia act as organelles for sensing and exploring the environment, as well as producing traction forces during cellular locomotion. Myosin-10 is a molecular motor crucial for intrafilopodial trafficking and filopodia formation. To decipher how myosin-10 generates force and movement, we used electron microscopy and a combination of ensemble biochemical and single molecule mechanical techniques to help elucidate its structure and mechano-chemical coupling. Our results (pp. E1833–E1842) clarify current controversies about myosin-10 structure and function by revealing that it generates an unexpectedly large biphasic power stroke and moves processively along actin, but detaches rapidly at relatively low force. These adaptations may be advantageous features for a myosin motor that carries bulky cargo within the narrow confines of the filopodium.
GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state
Erik C. Hansen, Kyle J. Seamon, Shannen L. Cravens, and James T. Stivers
The degradative dNTP triphosphohydrolase activity of the sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) enzyme helps maintain optimal dNTP balances for DNA replication and also serves as an HIV-1 restriction factor in resting CD4+ target cells of HIV by depleting dNTP substrates of reverse transcriptase. This study (pp. E1843–E1851) shows that full activation of SAMHD1 involves ordered binding of GTP and substrate dNTPs to activator and substrate sites on the enzyme, leading to ordered assembly of the tetramer active form. After the enzyme is activated, it no longer communicates with free activator nucleotides, which contributes to efficient depletion of dNTP pools in resting T cells.
A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1
Pierre Barraud, Silpi Banerjee, Weaam I. Mohamed, Michael F. Jantsch, and Frédéric H.-T. Allain
The double-stranded RNA-binding domain (dsRBD) is an abundant, conserved RNA-binding motif. Besides RNA binding, dsRBDs can serve as protein-interaction domains. In the human RNA-editing enzyme adenosine deaminase acting on RNA (ADAR1), one of its three dsRBDs mediates nuclear import by interacting with the import receptor transportin 1 (Trn1). RNA binding interferes with Trn1 binding, thereby preventing nuclear import. Using NMR spectroscopy and cell biological analysis, we show (pp. E1852–E1861) that the regions flanking this dsRBD form a bimodular Trn1-dependent nuclear localization signal. The dsRBD itself is not involved in Trn1 interaction but properly positions the Trn1 interacting regions. Using molecular modeling, we provide a structural explanation on how dsRNA binding prevents the dsRBD from accessing the interacting cavity of Trn1, thereby preventing nuclear import of RNA-bound ADAR1.
Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates
Harshad Ghodke, Hong Wang, Ching L. Hsieh, Selamawit Woldemeskel, Simon C. Watkins, Vesna Rapić-Otrin, and Bennett Van Houten
UV damage in genomic DNA is identified by the human UV-damaged DNA-binding protein (UV-DDB). Recognition of DNA damage by UV-DDB serves to initiate global genomic nucleotide excision repair (NER) in humans. Recent work has revealed that UV-DDB dimerizes at sites of damage. This study (pp. E1862–E1871) demonstrates that prior to stable damage recognition, UV-DDB interrogates DNA for damage via a 3D diffusion mechanism coupled to the formation of multiple transient intermediates. Stable binding at sites of damage is achieved by dimerization of UV-DDB. This study also analyzed a disease-causing mutant of UV-DDB, which was found to slide on DNA, while retaining the ability to dimerize on DNA. These results enhance our understanding of damage recognition in NER in humans.
Control of MT1-MMP transport by atypical PKC during breast-cancer progression
Carine Rossé, Catalina Lodillinsky, Laetitia Fuhrmann, Maya Nourieh, Pedro Monteiro, Marie Irondelle, Emilie Lagoutte, Sophie Vacher, François Waharte, Perrine Paul-Gilloteaux, Maryse Romao, Lucie Sengmanivong, Mark Linch, Johan van Lint, Graça Raposo, Anne Vincent-Salomon, Ivan Bièche, Peter J. Parker, and Philippe Chavrier
We characterize a mechanism through which the polarity protein atypical PKCι controls invasion and matrix remodeling by tumor cells by regulating endosome-to-plasma membrane traffic of the membrane type 1-matrix metalloproteinase (MT1-MMP) in breast-cancer cells. Further analysis shows that atypical PKCι and MT1-MMP are co–up-regulated in hormone receptor-negative breast tumors in association with higher risk of metastasis. These findings (pp. E1872–E1879) provide previously unidentified avenues for the design of therapeutic interventions.
Climate change, pink salmon, and the nexus between bottom-up and top-down forcing in the subarctic Pacific Ocean and Bering Sea
Alan M. Springer and Gus B. van Vliet
Wild salmon in the North Pacific Ocean, particularly pink salmon, have grown greatly since the mid-1970s apparently due to bottom-up effects of climate change on ocean physics and production processes. Pink salmon spend less than 2 y at sea and most stocks alternate between high and low levels of abundance every other year. In years of high abundance, they now constitute a pelagic consumer front as they return to their spawning rivers, exert top-down control over the open ocean ecosystem by outcompeting other species for shared prey resources, and drive major ecological shifts between years of high and low abundance (pp. E1880–E1888). Their effect on competing species must be considered in international conservation policies and when developing informed ecosystem-based management strategies.
Hypermutable DNA chronicles the evolution of human colon cancer
Kamila Naxerova, Elena Brachtel, Jesse J. Salk, Aaron M. Seese, Karen Power, Bardia Abbasi, Matija Snuderl, Sarah Chiang, Simon Kasif, and Rakesh K. Jain
Genetic heterogeneity in systemic cancer is of great clinical interest because it impacts therapeutic response and reflects how tumor cells grow and spread. We present (pp. E1889–E1898) a methodology that enables efficient evaluation of intratumor heterogeneity in patients through analysis of neutral somatic variation hotspots. Using only 20 genomic markers, we demonstrate a unique pattern of clonal diversity in each patient. Some tumors are significantly more diversified than others. Our data suggest that distinct clones can give rise to lymphatic and distant metastases. Our methodology is applicable to other human cancer types and facilitates high-throughput investigation of tumor evolution.
Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression
Yi-Jun Sheu, Justin B. Kinney, Armelle Lengronne, Philippe Pasero, and Bruce Stillman
During each cell-division cycle, eukaryotic cells initiate DNA synthesis from multiple replication origins on chromosomes to duplicate the entire genome once and only once. Spatial and temporal control of initiation and subsequent DNA synthesis at replication forks is important for maintaining genome integrity. Here we present a comprehensive analysis of patterns of origin activation, replication fork progression, and checkpoint responses in cells under replication stress. Our studies (pp. E1899–E1908) showed that a domain intrinsic to the replicative helicase, which unwinds DNA during replication, integrates multiple kinase-signaling pathways to control various aspects of the genome duplication process. Our work suggests a mechanism by which eukaryotic cells modulate the pattern of replication in response to environmental conditions through the replicative helicase.
Hassallidins, antifungal glycolipopeptides, are widespread among cyanobacteria and are the end-product of a nonribosomal pathway
Johanna Vestola, Tania K. Shishido, Jouni Jokela, David P. Fewer, Olli Aitio, Perttu Permi, Matti Wahlsten, Hao Wang, Leo Rouhiainen, and Kaarina Sivonen
New antifungal compounds are needed due to an increasing incidence of invasive fungal infections and resistance to many currently used drugs. Here (pp. E1909–E1917) we show that cyanobacteria are a rich source of antifungal compounds such as glycosylated lipopeptides, called hassallidins, which are commonly produced by filamentous nitrogen-fixing cyanobacteria. A diverse group of hassallidins and their complex nonribosomal biosynthesis were characterized in detail. Hassallidins and their previously unidentified biosynthetic enzymes offer new material for drug development. In addition, these compounds may have an ecological role in protecting cyanobacteria from parasitic fungi.
Automatic ultrarapid activation and inhibition of cortical motor systems in spoken word comprehension
Yury Shtyrov, Anna Butorina, Anastasia Nikolaeva, and Tatiana Stroganova
The mechanisms through which our brain generates complex cognitive percepts from simple sensory and motor events remain unknown. An important question is whether the basic brain structures controlling movements and perceptions directly participate in higher-order cognitive processes such as language comprehension. Using neurophysiology (pp. E1918–E1923), we found ultrarapid (starting at ∼80 ms) activations in the human motor cortex in response to unattended action-related verbs and nouns, with words related to different body parts activating corresponding body representations. Accompanying this category-specific activity was activation suppression by words with area-incompatible meaning, demonstrating operation of the neurophysiological principles of lateral/surround inhibition in language processing. These instant activations and deactivations emerging for words of different types in the absence of attention advocate automatic involvement of neural sensorimotor circuits in language comprehension.
Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells
Karen R. Kilcoyne, Lee B. Smith, Nina Atanassova, Sheila Macpherson, Chris McKinnell, Sander van den Driesche, Matthew S. Jobling, Thomas J. G. Chambers, Karel De Gendt, Guido Verhoeven, Laura O’Hara, Sophie Platts, Luiz Renato de Franca, Nathália L. M. Lara, Richard A. Anderson, and Richard M. Sharpe
Men are defined by androgens (testosterone), which drive fetal masculinization (male development) and puberty and maintain masculinity in adulthood, including sex drive, erectile function, and fertility. Moreover, Western cardiometabolic diseases are all associated with lowered testosterone levels in men. Therefore, influences on testosterone levels in adulthood have pervasive importance for masculinity and health. Our study shows (pp. E1924–E1932), for the first time, to our knowledge, that testosterone levels during fetal masculinization can (re)program adult testosterone levels through effects on stem cells, which develop into adult Leydig cells (the source of testosterone) after puberty. These stem cells are present in fetal testes of humans and animals, and using the latter, we show how these cells are reprogrammed to affect adult testosterone levels.