Polynucleotide kinase as a potential target for enhancing cytotoxicity by ionizing radiation and topoisomerase I inhibitors

Abstract

The cytotoxicity of many antineoplastic agents is due to their capacity to damage DNA and there is evidence indicating that DNA repair contributes to the cellular resistance to such agents. DNA strand breaks constitute a significant proportion of the lesions generated by a broad range of genotoxic agents, either directly, or during the course of DNA repair. Strand breaks that are caused by many agents including ionizing radiation, topoisomerase I inhibitors, and DNA repair glycosylases such as NEIL1 and NEIL2, often contain 5'-hydroxyl and/or 3'-phosphate termini. These ends must be converted to 5'-phosphate and 3'-hydroxyl termini in order to allow DNA polymerases and ligases to catalyze repair synthesis and strand rejoining. A key enzyme involved in this end-processing is polynucleotide kinase (PNK), which possesses two enzyme activities, a DNA 5'-kinase activity and a 3'-phosphatase activity. PNK participates in the single-strand break repair pathway and the non-homologous end joining pathway for double-strand break repair. RNAi-mediated down-regulation of PNK renders cells more sensitive to ionizing radiation and camptothecin, a topoisomerase I inhibitor. Structural analysis of PNK revealed the protein is composed of three domains, the kinase domain at the C-terminus, the phosphatase domain in the centre and a forkhead associated (FHA) domain at the N-terminus. The FHA domain plays a critical role in the binding of PNK to other DNA repair proteins. Thus each PNK domain may be a suitable target for small molecule inhibition to effectively reduce resistance to ionizing radiation and topoisomerase I inhibitors.

Figure 1

Common DNA lesions generated by exposure to ionizing radiation.

Figure 2

Schematic representation of DNA strand breaks induced by topoisomerase inhibitors and the role of PNK in the pathways responsible for their repair. Topo I inhibitors, such as camptothecin, produce strand breaks with a 5’-hydroxyl group and the enzyme covalently attached to a 3’-phosphate. Hydrolysis of the protein-DNA bond by tyrosyl-DNA phosphodiesterase (Tdp1) leaves a 3’-phosphate group. Therefore, both the 3’ and 5’ termini need to be acted upon by PNK. In contrast topo II inhibitors, such as etoposide, generate strand breaks with 3’-hydroxyl groups and the enzyme covalently linked to a 5’-phosphate. Although the mechanism(s) for repairing these lesions has yet to be fully elucidated, it is unlikely that PNK is required.

Figure 3

Processing of DNA strand break termini by PNK. PNK catalyzes the phosphorylation of 5’-hydroxyl (OH) termini and dephosphorylation of 3’-phosphate (P) termini so that subsequent nucleotide insertion and strand rejoining can be mediated by DNA polymerases and ligases, respectively.

Figure 4

Base excision repair, showing the importance of the AP lyase activities of the DNA glycosylases that act on a variety of base lesions. Under normal conditions, glycosylases that lack an AP lyase activity (e.g. MPG) or possess a β-elimination lyase activity (e.g. OGG1 and NTH1) depend on APE1 for the subsequent processing of the abasic sites. Glycosylases with a lyase acting by β.δ-elimination, such as NEIL1 and NEIL2, rely on PNK to remove the 3’-phosphate group. However, the lyase activity of NEIL1 and NEIL2 can also act on the intermediates generated by the other classes of DNA glycosylase, and therefore provide the basis for an alternative APE-independent repair pathway for these glycosylases. (Adapted from reference [30]).

Figure 5

Outline of the basic steps in the nonhomologous end-joining pathway for DNA double-strand break repair. PNK is required to process the strand break termini. In this pathway it interacts directly with phosphorylated XRCC4.

Figure 6

Structural overview of mammalian PNK. (A) Full-length mouse PNK. The FHA domain is shown in green, the phosphatase is in blue and kinase is in yellow. The catalytic Asp residues in the phosphatase and kinase (Asp 170 and Asp 396, respectively) are shown in pink. The P-loop in the kinase is shown in navy, and the sulfate ion bound in the P-loop is represented by red and orange spheres. The red arrow points to the phosphopeptide binding region of the FHA domain. (B) XRCC4 phosphopeptide (green) bound to the mouse PNK FHA. The superposition of 3 independent molecules of FHA in the crystal (A, B and C) is presented to illustrate the conformational variability of R44 due to crystal packing. The phosphopeptide from complex A is shown. (C) The kinase active site. The P-loop is shown in green with the bound sulfate (S1). Also shown is W1, the water molecule bound to the catalytic Asp 396 and a sulfate (S2) bound at the DNA binding site. W1 and S2 are proposed to mimic the substrate 5’-OH and a backbone phosphate, respectively. (D) A model of the minimal preferred kinase substrate bound to the kinase active site. The substrate is an 8-bp DNA duplex with a 5-nucleotide 3’ overhang. The PNK kinase domain is shown as a surface, colored by charge (positive in blue, negative in red). (E) The active site of mouse PNK phosphatase, overlaid with active sites of T4 PNK (purple, PDB ID 1LTQ)), phosphoserine phosphatase BeF₃/Mg²⁺ adduct (pink, PDB ID 1J97) and the phosphoaspartate form of β-phosphoglucomutase (green, PDB ID 1LVH). Residue numbering is for mPNK. (D) PNK phosphatase, in charged surface representation, with a modeled bound substrate, pC₄p).

Figure 7

Mechanism of 5’ DNA phosphorylation catalyzed by the PNK kinase domain. The role of the catalytic Asp 396 (Asp 397 in human PNK) is highlighted.

Figure 8

Mechanism of 3’ DNA dephosphorylation carried out by the PNK phosphatase domain. The mechanism is predicted to proceed via a covalent phospho-aspartate intermediate involving Asp 170. Putative transition states are indicated by ‡.

Figure 9

Scheme showing the assay for the kinase activity of PNK using a molecular beacon approach. Oligo A is 5’-phosphorylated by PNK and then annealed to the stem loop construct in which the fluorescence of the tetramethyl rhodamine dye (T) is quenched by the proximity of the dabcyl group (D). Ligation of oligo A to oligo B, which can only occur if oligo A is phosphorylated, forces the opening of the stem loop structure and enhancement of the fluorescence signal. (Adapted from reference [82]).