Structure-function studies of a plant tyrosyl-DNA phosphodiesterase provide novel insights into DNA repair mechanisms of Arabidopsis thaliana.
TDP1 (tyrosyl-DNA phosphodiesterase 1), a member of the PLD (phospholipase D) superfamily, catalyses the hydrolysis of a phosphodiester bond between a tyrosine residue and the 3'-phosphate of DNA. We have previously identified and characterized the AtTDP gene in Arabidopsis thaliana, an orthologue of yeast and human TDP1 genes. Sequence alignment of TDP1 orthologues revealed that AtTDP has both a conserved C-terminal TDP domain and, uniquely, an N-terminal SMAD/FHA (forkhead-associated) domain. To help understand the function of this novel enzyme, we analysed the substrate saturation kinetics of full-length AtTDP compared with a truncated AtTDP mutant lacking the N-terminal FHA domain. The recombinant AtTDP protein hydrolysed a single-stranded DNA substrate with Km and kcat/Km values of 703±137 nM and (1.5±0.04)×10(9) M(-1)·min(-1) respectively. The AtTDP-(Δ1-122) protein (TDP domain) showed kinetic parameters that were equivalent to those of the full-length AtTDP protein. A basic amino acid sequence (RKKVKP) within the AtTDP-(Δ123-605) protein (FHA domain) was necessary for nuclear localization of AtTDP. Analysis of active-site mutations showed that a histidine and a lysine residue in each of the HKD motifs were critical for enzyme activity. Vanadates, inhibitors of phosphoryl transfer reactions, inhibited AtTDP enzymatic activity and retarded the growth of an Arabidopsis tdp mutant. Finally, we showed that expression of the AtTDP gene could complement a yeast tdp1Δrad1Δ mutant, rescuing the growth inhibitory effects of vanadate analogues and CPT (camptothecin). Taken together, the results of the present study demonstrate the structure-based function of AtTDP through which AtTDP can repair DNA strand breaks in plants.
Department of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Hoyeun
H
Kim
Sang Hyeon
SH
Na
So-Young
SY
Lee
Young-Min
YM
Jeong
Hyun-Ju
HJ
Hwang
Jae Young
JY
Hur
Sang-Hyun
SH
Park
Je-Chang
JC
Woo
Sang-Gu
SG
Kim
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002166:Camptothecin / Q000494:pharmacology; D020134:Catalytic Domain; D002734:Chlorophyll / Q000378:metabolism; D004260:DNA Repair; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D014638:Vanadates / Q000494:pharmacology
D029681:Arabidopsis Proteins; D019913:Nuclear Localization Signals; D010726:Phosphodiesterase Inhibitors; D002734:Chlorophyll; D014638:Vanadates; D010727:Phosphoric Diester Hydrolases; C122147:tyrosyl-DNA phosphodiesterase; D002166:Camptothecin