HYBRIDOMA Volume 20, Number 4, 2001 Mary Ann Liebert, Inc. Production, Characterization, and Epitope Mapping of Monoclonal Antibodies Against Human Polydeoxyribonucleotide Kinase MESFIN FANTA, 1 HONG ZHANG, 1 NINA BERNSTEIN, 2 MARK GLOVER, 2 FERIDOUN KARIMI-BUSHERI, 1 and MICHAEL WEINFELD 1 ABSTRACT Polydeoxyribonucleotide kinase (PNK) is a mammalian DNA repair enzyme that has the capacity to phos- phorylate 59 DNA termini and dephosphorylate 39 DNA termini. A series of murine monoclonal antibodies (MAbs) was raised against the full-length recombinant human PNK. Seven of these antibodies were selected and characterized by enzyme immunoassay, Western blot analysis, and their capacity to immunoprecipitate PNK. The epitope location was defined by cyanogen bromide digestion and by using a truncated PNK for Western blot analysis. All of the MAbs recognize a single 60-kDa protein in human cell extracts. PNKs from calf, monkey, and Chinese hamster cell and tissue extracts were also detected by some or all of the MAbs. These antibodies can be successfully used for the cellular, biochemical, and functional analysis of PNK in dif- ferent mammalian cell lines. 237 INTRODUCTION P OLYNUCLEOTIDE KINASE (PNK) is an enzyme that acts at DNA strand break termini. It can phosphorylate DNA 59- hydroxyl termini and dephosphorylate 39-phosphate termini. (1) DNA strand breaks are frequently observed in cellular DNA. They occur during normal cellular activity, such as DNA repli- cation, recombination or differentiation, or through the action of endogenous or exogenous DNA damaging agents, such as reactive oxygen species and ionizing radiation. These strand breaks are associated with a variety of chemically distinct ter- mini, including 59-hydroxyl (2,3) and 39-phosphate groups. (2,4–6) But the pathways for strand repair require the presence of phos- phate and hydroxyl groups at the 59 and 39 termini, respectively, to facilitate DNA gap filling by a DNA polymerase and strand rejoining by a DNA ligase. (7,8) A 59-hydroxyl terminus can be converted to 59-phosphate either by the PNK-catalyzed trans- fer of the g-phosphate of ATP to the hydroxyl group, or by an exonuclease-mediated removal of the 59 terminal nucleoside. To date it is not known which is the predominant mechanism utilized by human cells. A number of mammalian enzymes are capable of hydrolyzing terminal 39-phosphate groups including the human AP endonuclease I (APE1) and its homologues, BAP (bovine) and APEX (murine). (7) However, when Habraken and Verly (9,10) purified 39-phosphatase activity from rat liver chro- matin, the major enzyme activity copurified with PNK. Work in our laboratory and others has provided strong evi- dence that PNK is indeed a DNA repair enzyme. We demon- strated that, unlike the well-known polynucleotide kinase coded by T4 phage, mammalian PNK acted efficiently at single-strand breaks and one nucleotide gaps in double stranded DNA, and together with DNA polymerase b and DNA ligase sealed the broken strand in such substrates. (11,12) Jilani et al. (13) observed that human PNK expressed in E. coli mutants was able to par- tially complement the absence of bacterial exonuclease III and endonuclease IV in response to DNA damage. Most recently, it has been shown that human PNK binds to other members of the base excision repair pathway including XRCC1, DNA poly- merase b, and DNA ligase III. (14) PNKs have been isolated and purified from several sources including rat liver and testes, calf thymus, and human HeLa cells. (15–20) Apart from some discrepancy in the molecular mass assigned to the polypeptides, they all show similar properties with respect to their kinase activity. All the proteins specifi- 1 Experimental Oncology, Cross Cancer Institute, Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Al- berta T6G 1Z2, Canada. 2 Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.