title: Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates.
abstract: Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, AB, Canada T6G 2H7.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Coquelle
authors.forename: Zahra
authors.initials: Z
authors.lastname: Havali-Shahriari
authors.forename: Nina
authors.initials: N
authors.lastname: Bernstein
authors.forename: Ruth
authors.initials: R
authors.lastname: Green
authors.forename: J N Mark
authors.initials: JN
authors.lastname: Glover
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D008958:Models, Molecular; D010710:Phosphates / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D010710:Phosphates; D004247:DNA; C530380:PNKP protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor); D045643:DNA Repair Enzymes