Highly Sensitive Radioactivity-Based DNA 3′-Phosphatase Activity Assay for Polynucleotide Kinase 3′-Phosphatase

Abstract

Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such lesions will impede transcription and replication and can lead to various cellular pathologies. Among various “dirty” DNA ends, 3′-phosphate is one of the most abundant lesions generated in the mammalian cells. Polynucleotide kinase 3′-phosphatase (PNKP) is the major DNA end-processing enzyme for resolving 3′-phosphate termini in the mammalian cells, and thus, it is involved in DNA base excision repair (BER), single-strand break repair, and classical nonhomologous end joining (C-NHEJ)-mediated DNA double-strand break (DSB) repair. The 3′-OH ends generated following PNKP-mediated processing of 3′-P are utilized by a DNA polymerase to fill in the gap, and subsequently, the nick is sealed by a DNA ligase to complete the repair process. Here we describe two novel assay systems to detect phosphate release by PNKP’s 3′-phosphatase activity and PNKP-mediated in vitro single-strand break repair with minimal repair components (PNKP, DNA polymerase, and DNA ligase) using either purified proteins or cell-free nuclear extracts from mammalian cells/tissues. These assays are highly reproducible and sensitive, and the researchers would be able to detect any significant difference in PNKP’s 3′-phosphatase activity as well as PNKP-mediated single-strand break repair activity in diseased mammalian cells/tissues vs normal healthy controls.