MOLECULAR-CLONING AND FUNCTIONAL-ANALYSIS OF THE ARABIDOPSIS-THALIANADNA-LIGASE-I HOMOLOG

A cDNA encoding the DNA ligase I homologue has been isolated from Arab idopsis thaliana using a degenerate PCR approach. The ORF of this cDNA encodes an amino acid sequence of 790 residues, representing a protei n with a theoretical molecular mass of 87.8 kDa and an isoelectric poi nt (pI) of 8.20. Alignment of the A. thaliana DNA ligase protein seque nce with the sequence of DNA ligases from human (Homo sapiens), murine (Mus musculus), clawed toad (Xenopus laevis) and the yeasts Schizosac charomyces pombe and Saccharomyces cerevisiae showed good sequence hom ology (42-45% identity, 61-66% similarity), particularly around the ac tive site. Sequence data indicate that the Arabidopsis DNA ligase is t he homologue of the animal DNA ligase I species. Functional analysis o f the cDNA clone demonstrated its ability to complement the conditiona l lethal phenotype of an S. cerevisiae cdc9 mutant defective in DNA li gase activity, confirming that the cloned sequence encodes an active D NA ligase. The level of the DNA ligase transcript was not increased in A. thaliana seedlings in response to DNA damage induced by a period o f enhanced UV-B irradiation. However, the cellular level of the DNA li gase mRNA transcript does correlate with the replicative state of plan t cells.