Award details

Proteomic approaches to a functional analysis of DNA ligases from Arabidopsis

Reference	BB/C502030/1
Principal Investigator / Supervisor	Dr Clifford Bray
Co-Investigators / Co-Supervisors	Dr Paul Sunderland, Professor David Thornton
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	257,078
Status	Completed
Type	Research Grant
Start date	01/01/2005
End date	30/04/2008
Duration	40 months

Abstract

Mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through control of translation initiation from either the first or second in-frame AUG codon respectively. We will quantify the effect of context in the vicinities of each start codon on translation initiation efficiency using (i) site direct mutagenesis of Kozak sequences around each start codon and quantification of the effects of these mutations in cell-free expression systems and (ii) confocal laser scanning microscopy of wt and mutated LIGI-GFP constructs expressed in planta. We will test the hypothesis that the presence of the mitochondrial presequence in AtLIG1 isoforms prevents the interaction of the AtLIG1 NLS with importin proteins resulting in hierarchical dominance of the presequence over the NLS in intracellular targeting. We will use both (i) the Y2H system and (ii) cell free expression systems with epitope tagged proteins to identify the presence/absence of importin-AtLIG1 isoform interactions. AtLIG1 interacting proteins which direct this ligase into specific pathways of DNA metabolism in planta will be identified using two complementary proteomic based approaches (i) a Y2H screen of an Arabidopsis cDNA library using the nuclear AtLIG1 isoform as bait and (ii) use of our AtLIG1 polyclonal antibody in pull down assays of Arabidopsis nuclear extracts from wt and the null lig4/6 double mutant followed by protein purifications and identification of specific proteins via MALDI-TOF MS. Fluorescence Resonance Energy Transfer (FRET) imaging microscopy of CFP and YFP fusion of AtLIG1 and putative interacting partner proteins expressed in transformed wt and double mutant Arabidopsis plants will be used to visualise interactions of partner proteins in planta in response to changes in developmental status and genome integrity of cells.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	Proteomics and Cell Function (PCF) [2003-2004]
Funding Scheme	X – not Funded via a specific Funding Scheme

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