Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates
Edward J. Bartlett, Nigel C. Brissett, and Aidan J. Doherty
Nonhomologous end joining (NHEJ) is a major DNA-break repair pathway in eukaryotes and prokaryotes but is assumed to be absent in archaea. This study (pp. E1984–E1991) establishes that a functionally homologous pathway is present in archaea. We have reconstituted archaeal NHEJ repair in vitro, demonstrating that it is closely related to the bacterial apparatus and preferentially repairs breaks using RNA intermediates. We identify a role for a functionally unascribed nuclease in preventing the accumulation of genotoxic repair intermediates produced by strand displacement. This study has important implications for our understanding of the mechanisms of DNA-break repair by NHEJ and the evolution of end-joining pathways.
Nuclease activity of Saccharomyces cerevisiae Dna2 inhibits its potent DNA helicase activity
Maryna Levikova, Daniel Klaue, Ralf Seidel, and Petr Cejka
The integrity of DNA must be preserved to pass genetic information onto the next generation and to prevent genomic instability. One of the key enzymes involved in DNA metabolism is the nuclease-helicase Dna2, which is required for both DNA replication and the repair of broken DNA. Our work (pp. E1992–E2001) revealed that Dna2 from Saccharomyces cerevisiae possesses a potent but cryptic helicase capacity, which is controlled by the nuclease activity of the same polypeptide. Regulating the interplay between both enzymatic activities might explain how Dna2 can take on its distinct cellular roles.
Distinct quaternary structures of the AAA+ Lon protease control substrate degradation
Ellen F. Vieux, Matthew L. Wohlever, James Z. Chen, Robert T. Sauer, and Tania A. Baker
Lon protease degrades unfolded or damaged proteins as well as numerous cellular regulatory proteins. How these different classes of substrates are recognized is poorly understood. We find (pp. E2002–E2008) that Lon hexamers assemble via a matrix of N-domain interactions to form a dodecamer with altered substrate-degradation properties. Access of protein substrates to the degradation machinery in the dodecamer appears to require passage through equatorial portals. As a consequence, large substrates that are efficiently degraded by hexamers seem to be preferentially excluded from dodecamers. This gating mechanism allows the substrate repertoire of Lon to be adjusted by its assembly state.
Dynamic CREB family activity drives segmentation and posterior polarity specification in mammalian somitogenesis
T. Peter Lopez and Chen-Ming Fan
The segmented axial skeleton of vertebrates is composed of an interlinked framework of vertebrae and ribs. During embryogenesis, vertebral precursors known as somites form sequentially from a progenitor tissue known as the presomitic mesoderm to foreshadow the metamerism of the axial skeleton. We have discovered (pp. E2019–E2027) that the cAMP responsive element binding protein (CREB) family of transcription factors operates in combination with Notch and Wnt signaling to instruct the timely scission of presomitic mesoderm into somites with proper anterior/posterior polarities. Thus, the CREB family represents a new and important molecular integrator in axial skeleton development. Our work has potential implications to spinal disorders such as scoliosis.
Molecular evolution of peptidergic signaling systems in bilaterians
Olivier Mirabeau and Jean-Stéphane Joly
Peptides and their specific receptors form molecular peptidergic systems (PSs) that are essential components of neuronal communication in the animal brain. Many PSs have been characterized in insects and mammals but their precise evolutionary relationship is not fully understood. We interrogated genomic sequence databases and used phylogenetic reconstruction tools to show that a large fraction of human PSs were already present in the last common ancestor of flies, mollusks, urchins, and mammals. Our analysis (pp. E2028–E2037) provides a comprehensive view of animal PSs that will pave the way for comparative studies leading to a better understanding of animal physiology and behavior.
Direct interaction between the TnsA and TnsB subunits controls the heteromeric Tn7 transposase
Ki Young Choi, Ying Li, Robert Sarnovsky, and Nancy L. Craig
DNA transposons move by excision and reintegration mediated by the element-encoded transposase. Unlike most transposases, the transposon Tn7 transposase is heteromeric, containing TnsB protein, which recognizes the transposon ends and mediates 3′ end breakage and joining, and TnsA protein, which mediates 5′ end breakage. We show that TnsA and TnsB interact and that TnsA can stimulate two key TnsB activities, end binding and end pairing. We also show that an end-pairing deficiency underlies the inability of a Tn7 containing two left ends to transpose. These findings (pp. E2038–E2045) reveal new aspects of protein–protein and protein–DNA interactions that underlie Tn7 transposition.
Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells
Daniela Weiskopf, Michael A. Angelo, Elzinandes L. de Azeredo, John Sidney, Jason A. Greenbaum, Anira N. Fernando, Anne Broadwater, Ravi V. Kolla, Aruna D. De Silva, Aravinda M. de Silva, Kimberly A. Mattia, Benjamin J. Doranz, Howard M. Grey, Sujan Shresta, Bjoern Peters, and Alessandro Sette
Dengue virus is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans, affecting over 100 million individuals each year. Currently there is no licensed vaccine or effective antiviral therapy available, and treatment is largely supportive in nature. This study (pp. E2046–E2053) presents a comprehensive analysis of functional T-cell memory against dengue viruses and suggests an HLA-linked protective role for CD8+ T cells. This demonstration of the protective role of T-cell responses points the way forward to identifying robust correlates of protection in natural immunity and vaccination against dengue virus.
Visualization of pinholin lesions in vivo
Ting Pang, Tinya C. Fleming, Kit Pogliano, and Ry Young
How a virus schedules the termination of its infection cycle is a mystery. Here (pp. E2054–E2063), we show that a small-membrane protein called a pinholin controls the infection cycle of a bacterial virus. By tagging the pinholin with a fluorescent marker, we show that, while the virus is replicating, the pinholin accumulates harmlessly and uniformly in the membrane until suddenly aggregating into foci (rafts), causing a fatal deenergization of the bacterial membrane and the onset of lysis. The entire temporal program of the virus may, thus, depend on achieving a critical concentration of a single protein.
Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling
Pawel Swietach, Jae-Boum Youm, Noriko Saegusa, Chae-Hun Leem, Kenneth W. Spitzer, and Richard D. Vaughan-Jones
The concentration of Ca2+ ions is kept low in cells by specialized ion-pumping proteins at the membrane. We show (pp. E2064–E2073) that in cardiac cells, cytoplasm also has an intrinsic ability to pump Ca2+. Histidyl dipeptides and ATP are diffusible cytoplasmic buffer molecules. By exchanging Ca2+ for H+, they act like local “pumps,” producing uphill Ca2+ movement within cytoplasm in response to H+ ion gradients. Intracellular H+ ions are generated locally by metabolism and competitively inhibit many Ca2+-activated biochemical processes. Recruiting Ca2+ to acidic zones facilitates these processes. Cytoplasmic histidyl dipeptides and ATP thus act like a biological pump without a membrane.
ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling
Miles A. Miller, Aaron S. Meyer, Michael T. Beste, Zainab Lasisi, Sonika Reddy, Karen W. Jeng, Chia-Hung Chen, Jongyoon Han, Keith Isaacson, Linda G. Griffith, and Douglas A. Lauffenburger
Regulated cell-surface proteolysis underpins processes of cellular migration in both physiological and pathological contexts. However, comprehending how multiple proteolytic events cohesively integrate to yield context-dependent cellular behavior remains a challenge. Here (pp. E2074–E2083) we present an experimental/computational paradigm for analyzing networks of protease activities that interface with signaling pathways to influence cellular migration in the invasive disease of endometriosis. We find that induced cellular migration is a quantitative consequence of positive feedback through ligand release and negative feedback through receptor shedding, which furthermore drives rapid resistance to kinase inhibitor treatment. Targeted clinical proteomics confirms dysregulated proteolysis in endometriosis.