Arabidopsis DNA double-strand break repair pathways.

DSBs (double-strand breaks) are one of the most serious forms of DNA damage that can occur in a cell's genome. DNA replication in cells containing DSBs, or following incorrect repair, may result in the loss of large amounts of genetic material, aneuploid daughter cells and cell death. There are two major pathways for DSB repair: HR (homologous recombination) uses an intact copy of the damaged region as a template for repair, whereas NHEJ (non-homologous end-joining) rejoins DNA ends independently of DNA sequence. In most plants, NHEJ is the predominant DSB repair pathway. Previously, the Arabidopsis NHEJ mutant atku80 was isolated and found to display hypersensitivity to bleomycin, a drug that causes DSBs in DNA. In the present study, the transcript profiles of wild-type and atku80 mutant plants grown in the presence and absence of bleomycin are determined by microarray analysis. Several genes displayed very strong transcriptional induction specifically in response to DNA damage, including the characterized DSB repair genes AtRAD51 and AtBRCA1. These results identify novel candidate genes that encode components of the DSB repair pathways active in NHEJ mutant plants.

Choice of a start codon in a single transcript determines DNA ligase 1 isoform production and intracellular targeting in Arabidopsis thaliana.

DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations. The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively. Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons. Wild-type AtLIG1-GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria. AtLIG1-GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus. Interestingly, the AtLIG1-GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria. This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4.

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane.

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.

Cloning and functional characterisation of a peptide transporter expressed in the scutellum of barley grain during the early stages of germination.

A peptide transport gene (HvPTR1) expressed in the scutella of germinating barley grain has been cloned by an RT-PCR approach. Sequence analysis of the full length cDNA (2260 bp) revealed an open reading frame encoding a 579 amino acid protein of predicted molecular mass 63 kDa, which displayed 58% identity to the Arabidopsis thaliana peptide transporter AtPTR2-B. Expression of HvPTR1 in Xenopus laevis oocytes conferred a 48-fold increase in alanyl-[14C]phenylalanine uptake relative to water injected oocytes, confirming the function of HvPTR1 as a peptide transporter. HvPTR1 expression was detectable only in the scutellum of the germinating barley grain, with no transcript found in roots, shoots or the embryo axis. Transcript levels increased rapidly from 6 to 24 h imbibition, correlating with the development of peptide transport activity in the barley scutellum. Peptide transport provides a significant source of organic nitrogen to the barley embryo for use in germination and growth processes associated with the early stages of seedling development. The temporal and spatial pattern of HvPTR1 expression is consistent with a central role for HvPTR1 in the transport of peptides in the germinating barley grain.