Identification of tyrosyl-DNA phosphodiesterase as a novel DNA damage repair enzyme in Arabidopsis

Tyrosyl-DNA phosphodiesterase (Tdp1) is a key enzyme which 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 been only described in yeast and human to date. In human, mutations of Tdp1 gene are involved in the disease spinocerebellar ataxia with axonal neuropathy (SCAN1). In plant, we have first identified the functional nuclear protein, AtTDP, homologue to human Tdp1 from Arabidopsis. The recombinant AtTDP protein certainly hydrolyzes the 3 ′ -phosphotyrosyl DNA substrates related to repairing of in vivo topoisomerase I-DNA induced damages. The loss-of-function AtTDP mutation displays developmental defects and dwarf phenotype in Arabidopsis. And this phenotype is substantially caused by decreased cell numbers without any changes of individual cell sizes. The tdp plants exhibit hypersensitivities to camptothecin (CPT), 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 for the repair of topoisomerase I-DNA complexes in Arabidopsis. and


INTRODUCTION
In all living organisms, a variety of DNA damages are constantly arisen by replication errors, UV, ionizing radiation, DNA damage agents, etc. Once DNA damage is occurred, specific DNA repair proteins, such as AP endonuclease, RAD1, RAD9, RAD51, XRCC2, Ku80, ligase, etc., initiate to act through the repair pathways (Wood et al., 2001). Defects in DNA damage repair have evolved into the cancer or genetic disease in mammals and affect productivity or growth in plants (Tuteja et al., 2001;Wood et al., 2001).
In repair of DNA-protein crosslinks, tyrosyl-DNA phosphodiesterase 1 (Tdp1) is known as unique protein.
Tdp1 protein was initially reported as an active enzyme in Saccharomyces cerevisiae that specifically removes tyrosine group from the covalent intermediate between the tyrosine residue and the terminal 3´-phosphate of the oligonucleotide (Yang et al., 1996). Subsequently, yeast TDP1 gene was identified and showed highly conserved sequences with other organisms, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Homo sapiens (Pouliot et al., 1999). The Tdp1 homologues of these species are a member of phospholipase D (PLD) superfamily (Pouliot et al., 1999;Interthal et al., 2001). Yeast Tdp1 is mainly studied about topoisomerase Irepair pathway by 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, 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 been resulted in 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 was caused by the missense mutation (His493Arg) in the Tdp1 catalytic site. As in yeast, 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 (SSB) and double strand break 1 0

DISCUSSION
We have identified Arabidopsis TDP gene, human and yeast tyrosine-DNA phosphodiesterase (Tdp1) homologues, related to the repair of covalent protein-DNA adducts. Arabidopsis TDP has 34% sequence identity with H. sapiens and 43% identity with S. cerevisiae. Tdp1 proteins in yeast and human, as a member of PLD superfamily, show the HKD signature motif which is in an active site of Tdp1 enzyme (Interthal et al., 2001). Even though Arabidopsis TDP shows the low sequence homology to those of human and yeast, AtTDP protein remarkably contains two conserved HKD motifs needed for enzyme activity of TDP that specifically hydrolyzes the topoisomerase I-DNA complexes. And the protein is efficiently localized in nucleus. Therefore, we supposed the possibility that AtTDP protein may show the enzyme activity in a similar manner as yeast and human Tdp1 proteins. In biochemical and functional analysis, the recombinant AtTDP protein was certainly active on single stranded, tailed, and blunted duplex DNAs, catalyzing the hydrolysis of 3′-phosphotyrosine bond and the substrate preferences of AtTDP are well consistent with those of yeast and human Tdp1s.
The novel AtTDP protein is required for normal growth and development in Arabidopsis. Plant homozygous for loss-of-function AtTDP mutation resulted in dwarf phenotype with developmental defects in Arabidopsis.
The abnormalities of tdp plants were observed in vegetative and flowering development, showing reduced fertility. From the early vegetative stage, growth was retarded and organ size was very small in tdp mutants.
Moreover, stems of tdp plants were very slender. The flowers were a quite small and displayed very low fertility.
To understand the causes of decreased organ size in tdp plants, we analyzed some parameters in detail. As in results, while the organ sizes of leaves and petals in tdp mutants were largely reduced as compared with those of wild-type plants, the epidermal cell sizes in leaves and petals were unchanged in tdp mutants. Therefore, we analyzed whether the mitotic cell cycle genes were expressed in tdp mutants. CYCD3:1 and CDKA have an important role in G1-to-Sphase transition (Menges et al., 2006). A nonmitotic cyclin, CYCD3:1 gene plays the integration of cell division in Arabidopsis leaf. At a DNA damage checkpoint, CDKA stops the progression of the cell cycle when DNA is damaged. Moreover, CDKA was reported as a target of WEE1 kinase in Arabidopsis. The Arabidopsis wee1 gene decreases cell division related to cell cycle arrest on the DNA integrity checkpoints (De Schutter et al., 2007). The expression of mitotic cell cycle related genes did not differ significantly between wild-type and tdp plants. These results indicate that AtTDP did not affect genes that are involved in the control of cell cycle. Therefore, we further examined the possibility that decreased cell number 1 1 in tdp plants is caused by progressive cell death during Arabidopsis development. Indeed, our sensitivity test to DNA damage agents revealed that tdp plants were hypersensitive to CPT. In addition, CPT-induced cell death was intensively observed in rosette leaves of tdp plants than in wild-type, suggesting the failure of DNA damage repair and progressive cell death in tdp mutant. To determine whether mutant plants are generated by cell death, we directly stained cotyledons and rosette leaves of wild-type and tdp plants with trypan blue. The tdp plants strongly showed progressive cell death from early developmental stage. These results indicate that accumulation of DNA damage caused by the loss-of AtTDP function induced cell death in Arabidopsis. CPT-induced cell death was also observed in yeast and human tdp1 mutants. The Human Tdp1 gene in SCAN1 cells is hypersensitive to CPT and induces the cell death in treated cells (El-Khamisy et al., 2005;Interthal et al., 2005).
Yeast tdp1 mutants, when coupled with mutation of the other DNA repair genes, show a significant effect on survival of CPT-treated cells (Pouliot et al., 2001;Liu et al., 2002;Vance and Wilson, 2002;Liu et al., 2004) .
All together our data demonstrate that the correlation between the tdp mutant phenotype and biochemical function of AtTDP protein is well consistent with Tdp1's role in related to repairs of topoisomerase I-induced damages. Therefore, we conclude that failure in repair of topoisomerases-mediated DNA damage could be accumulation of these products in the cells and to cell death that results in the dwarf phenotype during Arabidopsis development. This finding provides a better understanding of role of AtTDP during Arabidopsis development.  , 1995). T-DNA border-specific primers (AtLB1, AtLB2, and AtLB3) and a pool of two arbitrary degenerate primers (DEG1, DEG2) were used for three rounds of TAIL-PCR cycling (Supplemental Table S2). Homozygous tdp was selected by BASTA segregation analysis and verified by PCR.

Identification and characterization of the mutant
For complementation, the cDNA of AtTDP was amplified using RT-PCR with specific primers (AtTDP-F and www.plantphysiol.org on August 21, 2017 -Published by Downloaded from Copyright © 2010 American Society of Plant Biologists. All rights reserved. Table S2). The cDNA fragment containing AtTDP open reading frame was cloned into pGEM-T Easy vector (Promega). The positive plasmid was subcloned into the binary vector pBI121 (CLONTECH). An identified positive clone was used for transformation of Agrobacterium tumefaciens C58C1 by the heat-shock method. And the plasmids were transformed into Arabidopsis tdp plants using floral dip method (Clough and Bent, 1998). We obtained the independent transgenic lines with kanamycin and BASTA resistances.

AtTDP-R) (Supplemental
All Arabidopsis plants were grown in long days (16h light/8h dark) under fluorescent lights at 22°C with 70% humidity.

Nuclear localization of AtTDP-GFP fusion protein
To make a AtTDP-GFP fusion protein, the AtTDP cDNA sequence was amplified by PCR using the G-F and G-R primers containing a BamHI site and fused to the GFP (Supplemental Table S1). Rosette leaves of wildtype plants grown for 2 weeks were used for the isolation and the transformation of protoplasts. Ten μg of plasmid DNAs containing AtTDP-GFP fusion constructs was transfected into protoplasts. Then, protoplasts were incubated in a dark condition at 24°C for 24 h. Images were obtained using a confocal microscope (BIO-RAD, Radiance 2000/MP).  S1). The substrate-change assay was examined in the same manner as the enzyme activity assay.

Histology and Microscopy
To obtain the cross-sections of stems, samples were placed in a fixation solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) under vacuum conditions for 2 days at 4°C. Fixed samples were rinsed with phosphate buffer twice and dehydrated through a series of graded ethanol. After infiltration with xylene, samples were embedded in paraplasts. Eight μ 1 5

Cell death measurement
The leaves and cotyledons of wild-type and tdp plants were boiled for 1.5 min in the solution prepared as follow: phenol: lactic acid: glycerol: water: 95% ethanol at the ratio of 1:1:1:1:4 with 0.4% (w/v) trypan blue (Invitrogen). Samples were destained using 0.1% chloral hydrate (Sigma-Aldrich) for 2 days and then mounted in 50% glycerol (Koch and Slusarenko, 1990). The treated plants were imaged with a Nikon COOLPIX 4500 digital camera.

Acknowledgements
We wish to express our sincere thanks to Prof. Donald J. Armstrong (Oregon State University, Corvallis, OR, USA) for helpful discussion and critical review.    P  o  u  l  i  o  t  J  J  ,  R  o  b  e  r  t  s  o  n  C  A  ,  N  a  s  h  H  A   (  2  0  0  1  )  P  a  t  h  w  a  y  s  f  o  r  r  e  p  a  i  r  o  f  t  o  p  o  i  s  o  m  e  r  a  s  e  I  c  o  v  a  l  e  n  t   c  o  m  p  l  e  x  e  s  i  n  S  a  c  c  h  a  r  o  m  y  c  e  s  c  e  r  e  v  i  s  i  a  e  .  G  e  n  e  s  C  e  l  l  s   6  :   6  7  7  -6  8  7   P  o  u  l  i  o  t  J  J  ,  Y  a  o  K  C  ,  R  o  b  e  r  t  s  o  n  C  A  ,  N  a  s  h  H  A   (  1  9  9  9  )  Y  e  a  s  t  g  e  n  e  f  o  r  a  T  y  r  -D  N  A  p  h  o  s  p  h  o  d  i  e  s  t  e  r  a  s