Abstract
BackgroundDNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. Eukaryotes possess multiple DNA ligases with distinct roles in DNA metabolism, with clear differences in the functions of DNA ligase orthologues between animals, yeast and plants. DNA ligase 1, present in all eukaryotes, plays critical roles in both DNA repair and replication and is indispensable for cell viability.ResultsKnockout mutants of atlig1 are lethal. Therefore, RNAi lines with reduced levels of AtLIG1 were generated to allow the roles and importance of Arabidopsis DNA ligase 1 in DNA metabolism to be elucidated. Viable plants were fertile but displayed a severely stunted and stressed growth phenotype. Cell size was reduced in the silenced lines, whilst flow cytometry analysis revealed an increase of cells in S-phase in atlig1-RNAi lines relative to wild type plants. Comet assay analysis of isolated nuclei showed atlig1-RNAi lines displayed slower repair of single strand breaks (SSBs) and also double strand breaks (DSBs), implicating AtLIG1 in repair of both these lesions.ConclusionReduced levels of Arabidopsis DNA ligase 1 in the silenced lines are sufficient to support plant development but result in retarded growth and reduced cell size, which may reflect roles for AtLIG1 in both replication and repair. The finding that DNA ligase 1 plays an important role in DSB repair in addition to its known function in SSB repair, demonstrates the existence of a previously uncharacterised novel pathway, independent of the conserved NHEJ. These results indicate that DNA ligase 1 functions in both DNA replication and in repair of both ss and dsDNA strand breaks in higher plants.
Highlights
DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways
Phenotypic analyses of DNA ligase1 deficient plants In the absence of viable knockout lines, Arabidopsis plants with reduced levels of ligase 1 (LIG1) were generated using an RNAi approach to gain further insight into gene function (Figure 1A). Both Arabidopsis DNA LIGASE 1 (AtLIG1) transcript and protein levels in the silenced lines were determined by semi-quantitative RT-PCR and Western blotting respectively (Figures 1B and 1C)
Two lines with reduced levels of Arabidopsis DNA ligase I (AtLIG1) protein were selected for further analysis and designated atlig1-RNAiA and atlig1-RNAiB
Summary
DNA ligase enzymes catalyse the joining of adjacent polynucleotides and as such play important roles in DNA replication and repair pathways. DNA ligases play essential roles in all organisms by maintaining the physical structure of DNA These enzymes seal gaps in the sugar-phosphate backbone of DNA that arise during DNA replication, DNA damage and repair. DNA ligase 1 plays important roles in DNA single strand break (SSB) repair pathways in mammals and yeast. These pathways are less well characterised in plants, but orthologues of several SSB repair genes are identifiable in the genomes of higher plants [5]. Whilst LIG1 is essential for cell division in yeast and plants, mouse embryos are viable and develop until mid-term without LIG1, indicating that a second ligase may substitute for growth up to this point [9]. Plants deficient in AtLIG1 are null, cell division in gametophytes prior to fertilisation appeared unaffected, suggesting that either that a second ligase can partially substitute for DNA ligase 1, or that ligase 1 levels in haploid cells are sufficient to support gametogenesis [8]
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