Identification of DNA 3'-phosphatase active site residues and their differential role in DNA binding, Mg2+ coordination, and catalysis.
DNA 3'-phosphatase (Tpp1) from Saccharomyces cerevisiae, a homologue of human polynucleotide kinase/3'-phosphatase, has been shown to participate in DNA damage repair by removing 3'-phosphate blocking lesions. Tpp1 shows similarity to the l-2-haloacid dehalogenase superfamily of enzymes. By comparison to phosphoserine phosphatase, a well-studied member of this family, we designed conservative and nonconservative substitutions of likely active site residues of Tpp1 and tested them in a variety of assays. From the loss or impairment of activity, we identified D35, D37, T39, S88, K170, D206, and D218 as being involved in Tpp1 catalysis. D35 and K170 were the most critical since maximum inactivation was seen with even conservative mutations. Tpp1 bound DNA through its active site in a Mg(2+)-dependent manner and exhibited a preference for dsDNA. Although Tpp1 bound more strongly to DNA with a free 3' terminus, it also bound well to covalently closed DNA, suggesting a possible lesion scanning mechanism. DNA binding studies further indicated that Tpp1 coordinates Mg(2+) through D35 and D206 and contacts the DNA 3' end through D37. The removal of 3'-phosphate involved a phospho-Tpp1 intermediate, and our results support D35 as being the point of covalent attachment. On the basis of these similarities in mutant phenotypes of Tpp1 and phosphoserine phosphatase, we propose a reaction mechanism for Tpp1 which explains its strict phosphate specificity.
Deshpande
Rajashree A
RA
Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, M4214 Medical Sciences Building I, Box 0602, Ann Arbor, Michigan 48109-0602, USA.
Wilson
Thomas E
TE
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009708:Nucleotidases / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000091346:Tripeptidyl-Peptidase 1
D009841:Oligonucleotides; D000091346:Tripeptidyl-Peptidase 1; D004247:DNA; D005982:Glutathione Transferase; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase; C000717267:TPP1 protein, human; D008274:Magnesium