Molecular Cloning of the Human Gene, PNKP, Encoding a Polynucleotide Kinase 3′-Phosphatase and Evidence for Its Role in Repair of DNA Strand Breaks Caused by Oxidative Damage

Arshad Jilani,Dindial Ramotar,Carolyn Slack,Colin Ong,Xiao Ming Yang,Stephen W Scherer,Dana D Lasko

doi:10.1074/jbc.274.34.24176

Abstract

Mammalian polynucleotide kinases catalyze the 5'-phosphorylation of nucleic acids and can have associated 3'-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5'-DNA kinase and 3'-phosphatase activities identified human and murine dbEST clones. The 57.1-kDa conceptual translation product of this gene, polynucleotide kinase 3'-phosphatase (PNKP), contained a putative ATP binding site and a potential 3'-phosphatase domain with similarity to L-2-haloacid dehalogenases. BLAST searches identified possible homologs in Caenorhabditis elegans, Schizosaccharomyces pombe, and Drosophila melanogaster. The gene was localized to chromosome 19q13.3-13.4. Northern analysis indicated a 2-kilobase mRNA in eight human tissues. A glutathione S-transferase-PNKP fusion protein displayed 5'-DNA kinase and 3'-phosphatase activities. PNKP is the first gene for a DNA-specific kinase from any organism. PNKP expression partially rescued the sensitivity to oxidative damaging agents of the Escherichia coli DNA repair-deficient xth nfo double mutant. PNKP gene function restored termini suitable for DNA polymerase, consistent with in vivo removal of 3'-phosphate groups, facilitating DNA repair.

Highlights

Mammalian polynucleotide kinases catalyze the 5؅phosphorylation of nucleic acids and can have associated 3؅-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage
Identification of dbEST Clones Containing Peptide Sequence from the Bovine DNA Kinase SNQI-polynucleotide kinase (PNK)—Two bovine peptide sequences were found from analysis of method 2 purified material, I/LVI/LFTN[Q/K]MGI/LGR, and I/LI/LYI/ LEI/L(PR) where I/L or Q/K symbolizes an isobaric amino acid, the brackets indicate an ambiguous residue assigned according to the sequence found in a dbEST hit, and the parentheses indicate residues assigned with uncertainty
Human polynucleotide kinase 3؅-phosphatase (PNKP) Acts in Vivo to Process H2O2-induced 3ЈBlocking DNA Lesions—To directly test whether PNKP is acting in vivo to remove 3Ј-blocking DNA lesions at ssb, we examined if chromosomal DNA isolated from H2O2-treated cells could sustain in vitro DNA repair synthesis by E. coli DNA polymerase I [39]

Summary

Introduction

Mammalian polynucleotide kinases catalyze the 5؅phosphorylation of nucleic acids and can have associated 3؅-phosphatase activity, predictive of an important function in DNA repair following ionizing radiation or oxidative damage. The sequences of three tryptic peptides from a bovine 60-kDa polypeptide that correlated with 5؅-DNA kinase and 3؅-phosphatase activities identified human and murine dbEST clones.

Results

Conclusion