The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP.P(i) being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P(i) release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se

Crop Updates 2009 - Genetically Modified Crops, Nutrition, Soils, & Others

This session covers fifteen papers from different authors: 1. Performance of Canola Breeders Roundup Ready® canola hybrid CHYB-166 in 2008, Wallace Cowling, Canola Breeders Western Australia Pty Ltd 2. The implications of GM glyphosate resistant lupin, Art Diggle, Caroline Peek, Frank D’Emden, Fiona Evans, Bob French, Rob Grima, Sam Harburg, Abul Hashem,, John Holmes, Jeremy Lemon, Peter Newman, Janet Paterson, Steve Penny,Department of Agriculture and Food, Peter Portmann, Agriconnect 3. Nufarm Roundup Ready® Canola Systems Trials— 2008 Mark Slatter, Research and Development Officer, Victoria, Nufarm (0438 064 845) Angus MacLennan, Business Development Manager, New South Wales, Nufarm (0408 358 024) Cooperators: Monsanto, Nuseed, Pacific Seeds, Pioneer Seeds 4. Roundup Ready® canola—2008 Limited Commercial Release. Getting the system right, Andrew Wells and Mark Slatter, Nufarm Australia Limited (Reprint from 2008 GRDC Cropping Updates with Introductory note) NUTRITION 5. Fertilising in a changing price environment, Bill Bowden1, Wayne Pluske2 and Jeremy Lemon1, 1Department of Agriculture and Food, 2Back Paddock Company 6. Making better fertiliser for Western Australian cropping systems, Wen Chen1 2, Geoff Anderson1, Ross Brennan1and Richard Bell2 1Department of Agriculture and Food, 2School of Environmental Science, Murdoch University 7. The nitrogen fertiliser replacement value of biosolids from wastewater treatment, Hannah Rigby1, Deborah Pritchard1, David Collins1, Katrina Walton2, David Allen2 and Nancy Penney31School of Agriculture and Environment,Curtin University of Technology, Muresk Campus, 2Chemistry Centre of Western Australia 3Water Corporation of Western Australia 8. Fertilising to soil type (usually) pays, Michael Robertson, Bill Bowden and Roger Lawes, CSIRO, Floreat and Department of Agriculture and Food SOILS 9. Management of subsoil acidity and compaction using a combination of lime, deep ripping and controlled traffic, Stephen Davies, Chris Gazey, Breanne Best and David Gartner, Department of Agriculture and Food 10. Optimising gypsum applications through remote sensing and Variable Rate Technology, Frank D’Emden, Department of Agriculture and Food and Quenten Knight,Precision Agronomics Australia 11. Case study of a 17 year agricultural lime trial, Chris Gazey1, Joel Andrew2and Ryan Pearce3 1Department of Agriculture and Food; 2Precision SoilTech; 3ConsultAg 12. Soil organic carbon in WA agricultural soils, FC Hoyle and A Bennett, Department of Agriculture and Food OTHER 13. Is the no-till revolution complete in WA? Frank D’Emden1, Rick Llewellyn2 and Ken Flower3 1Department of Agriculture and Food, 2CSIRO Sustainable Ecosystems, 3University of Western Australia 14. Progression Planning (The Concept), Julian Krieg and Owen Catto, Wheatbelt Men’s Health 15. Is the Department of Agriculture and Food still a primary source of cropping information? Cindy Parsons, Department of Agriculture and Foo

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.This work was supported by funding from the Medical Research Council and the European Research Council (ERC, 281847) (A.P.J.), the Lister Institute for Preventative Medicine (A.P.J. and G.S.S.), Medical Research Scotland (L.S.B.), German Federal Ministry of Education and Research (BMBF, 01GM1404) and E-RARE network EuroMicro (B.W), Wellcome Trust (M. Hurles), CMMC (P.N.), Cancer Research UK (C17183/A13030) (G.S.S. and M.R.H), Swiss National Science Foundation (P2ZHP3_158709) (O.M.), AIRC (12710) and ERC/EU FP7 (CIG_303806) (S.S.), Cancer Research UK (C6/A11224) and ERC/EU FP7 (HEALTH-F2- 2010-259893) (A.N.B. and S.P.J.).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ng.345