ABSTRACT
Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks.
Subject(s)
Nucleotidases/chemistry , Nucleotidases/metabolism , Plasmodium falciparum/enzymology , Polynucleotide 5'-Hydroxyl-Kinase/chemistry , Polynucleotide 5'-Hydroxyl-Kinase/metabolism , Amino Acid Sequence , Enzyme Stability , Humans , Molecular Sequence Data , Nucleotidases/genetics , Plasmodium falciparum/chemistry , Plasmodium falciparum/genetics , Polynucleotide 5'-Hydroxyl-Kinase/genetics , Protein Structure, Tertiary , Sequence Alignment , Substrate SpecificityABSTRACT
The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Nuclear Factor 45 Protein/metabolism , Nuclear Factor 90 Proteins/metabolism , Nuclear Proteins/metabolism , Nucleoproteins/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Phosphotransferases/metabolism , Protein Phosphatase 1/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Binding Sites/genetics , Conserved Sequence/genetics , HeLa Cells , Humans , Molecular Chaperones , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nucleoproteins/genetics , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Phosphotransferases (Alcohol Group Acceptor)/genetics , Protein Binding/genetics , Protein Phosphatase 1/chemistry , Protein Subunits/chemistry , Protein Subunits/metabolism , Rabbits , Rats , Repressor ProteinsABSTRACT
Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.