Identification of Tyrosyl-DNA Phosphodiesterase as a Novel DNA Damage Repair Enzyme in Arabidopsis

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a key enzyme that hydrolyzes the phosphodiester bond between tyrosine of topoisomerase and 3′-phosphate of DNA and repairs topoisomerase-mediated DNA damage during chromosome metabolism. However, functional Tdp1 has only been described in yeast and human to date. In human, mutations of the Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy. In plants, we have identified the functional nuclear protein AtTDP, homolog to human Tdp1 from Arabidopsis (Arabidopsis thaliana). The recombinant AtTDP protein certainly hydrolyzes the 3′-phosphotyrosyl DNA substrates related to repairing in vivo topoisomerase I-DNA-induced damage. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. This phenotype is substantially caused by decreased cell numbers without any change of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin, a potent topoisomerase I inhibitor, and show rigorous cell death in cotyledons and rosette leaves, suggesting the failure of DNA damage repair in tdp mutants. These results indicate that AtTDP plays a clear role in the repair of topoisomerase I-DNA complexes in Arabidopsis.In all living organisms, a variety of DNA damage is constantly caused by replication errors, UV light, ionizing radiation, DNA damage agents, etc. Once DNA damage has occurred, specific DNA repair proteins, such as AP endonuclease, RAD1 (for radiation sensitive), RAD9, RAD51, XRCC2 (for x-ray repair cross-complementing), Ku80 (XRCC6), and ligase, initiate to act through the repair pathways (Wood et al., 2001). Defects in DNA damage repair have evolved into cancer or genetic diseases in mammals and affect productivity or growth in plants (Tuteja et al., 2001; Wood et al., 2001).In the repair of DNA-protein cross-links, tyrosyl-DNA phosphodiesterase 1 (Tdp1) is known as a unique protein. Tdp1 was initially reported as an active enzyme in Saccharomyces cerevisiae that specifically removes the Tyr group from the covalent intermediate between the Tyr residue and the terminal 3′- phosphate of the oligonucleotide (Yang et al., 1996). Subsequently, the yeast TDP1 gene was identified and showed highly conserved sequences with other organisms, such as Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (Pouliot et al., 1999). The Tdp1 homologs of these species are members of the phospholipase D (PLD) superfamily (Pouliot et al., 1999; Interthal et al., 2001). Yeast Tdp1 is mainly studied concerning the topoisomerase I-repair pathway using double or triple mutants. The deletion mutations of yeast Tdp1 were shown lacking in the repair of DNA damage induced by a topoisomerase inhibitor, the anticancer drug camptothecin (CPT; Pouliot et al., 2001; Liu et al., 2002; Vance and Wilson, 2002). Tdp1 has been further implicated in multiple repair pathways, including the damage repair of topoisomerase II-DNA in yeast (Nitiss et al., 2006).In multicellular eukaryotes, the defect of human Tdp1 has resulted in the neurodisorder disease spinocerebellar ataxia with axonal neuropathy (SCAN1; Takashima et al., 2002). SCAN1 is a rare autosomal recessive neurodegenerative disease, and the patients present distal muscle weakness and peripheral neuropathy (Interthal et al., 2001; Takashima et al., 2002). SCAN1 is caused by a missense mutation (His-493Arg) in the Tdp1 catalytic site. As in yeast, the human Tdp1 protein plays a role in the repair of topoisomerase I-DNA complex lesions in SCAN1 cells (El-Khamisy et al., 2005; Miao et al., 2006). SCAN1 cells are hypersensitive to CPT (Interthal et al., 2005; Miao et al., 2006) and accumulate single-strand break and double-strand break DNAs by CPT (El-Khamisy et al., 2005).At present, although the functional analysis of Tdp1 has been widely conducted in yeast and human cell lines, its role in the overall growth and development of higher plants remains unknown. Here, we investigate the function of a novel Arabidopsis (Arabidopsis thaliana) TDP, a human and yeast Tdp1 homolog. The AtTDP protein shows the DNA damage-repairing activity and substrate specificities in biochemical assay. The dwarf phenotype of the Arabidopsis tdp mutant may be due to the reduced cell number caused by the accumulation of DNA damage and progressive cell death during Arabidopsis development.