Identification of DNA 3'-phosphatase active site residues and their differential role in DNA binding, Mg2+ coordination, and catalysis

Biochemistry
Rajashree A Deshpande, Thomas E Wilson

Abstract

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 t...Continue Reading

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Citations

Jul 10, 2010·Proceedings of the National Academy of Sciences of the United States of America·Pravin A NairStewart Shuman
Dec 21, 2011·Proceedings of the National Academy of Sciences of the United States of America·Matthew J Schellenberg, R Scott Williams
Sep 4, 2009·The Journal of Biological Chemistry·Tapan BiswasOleg V Tsodikov
Feb 5, 2009·Nucleic Acids Research·Moritz NiemannH Ulrich Göringer
Oct 12, 2010·Nucleic Acids Research·Karim BahmedJohn L Nitiss
Aug 10, 2010·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Lisandro H OteroArmando Albert
Dec 24, 2013·Biochemistry·Gérald Larrouy-MaumusLuiz Pedro Sório de Carvalho
Nov 1, 2015·Nucleic Acids Research·Nicholas C BauerPaul W Doetsch
Mar 12, 2010·Protein Science : a Publication of the Protein Society·Mengmeng ZhangYan Zhang
Mar 8, 2005·Molecular Cell·Nina K BernsteinJ N Mark Glover
Sep 13, 2006·The Journal of Biological Chemistry·Erumbi S RangarajanMiroslaw Cygler
Jan 21, 2013·Journal of Materials Chemistry. B, Materials for Biology and Medicine·Wenping ZhuRuqin Yu
Sep 19, 2018·Current Genetics·Sarah R Offley, Martin C Schmidt

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