The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.

Mammalian cells contain potent activity for removal of 3-phosphoglycolates from single-stranded oligomers and from 3 overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a singlestranded 3 terminus to leave a 3-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well as Saccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3-phosphoglycolate on a 3-base 3 overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3 overhangs.Ionizing radiation, radiomimetic drugs, and to some extent all free radical-based genotoxins induce DNA double strand breaks (DSBs) 1 by oxidative fragmentation of DNA sugars (1-4). Most such breaks bear terminal 3Ј-phosphate or 3Ј-phosphoglycolate (PG) moieties (1, 2, 5) that must be removed to allow fill-in of gaps by DNA polymerase and final religation by DNA ligase. While the human apurinic/apyrimidinic endonuclease Ape1 can remove PGs, albeit inefficiently, from blunt and recessed 3Ј ends of DSBs (6), PGs on 3Ј overhangs are highly resistant to this enzyme (6, 7). Nevertheless, in mammalian cell extracts, PGs on 3Ј overhangs of DSBs are removed by an as yet unidentified activity, and a fraction of such DSBs are accurately rejoined by Ku-mediated end alignment, gap-filling, and ligation (8).The yeast tyrosyl-DNA phosphodiesterase scTdp1 was isolated from Saccharomyces cerevisiae as an activity that hydrolyzes the phosphodiester bond linking tyrosine to a 3Ј DNA end (9). Such linkages are formed as intermediates in DNA relaxation by topoisomerase I and in DNA cleavage and reunion by various recombinases. These intermediates can become trapped if the process is interrupted, for example, by a topoisomerase inhibitor or by collision with a replication fork. The hypersensitivity of tdp1⌬ yeast to topoisomerase I-mediated DNA damage (10) suggests a critical role for scTdp1 in repair of such lesions. A human homologue with similar 3Ј-phosphotyrosyl-processing activity, hTdp1, was cloned by homology to scTdp1 and was eventually shown to have some homology to the phospholipase D superfamily of phosphodiesterases (11). Yeast and human Tdp1 differ from other activities for processing blocked 3Ј ends in that they leave a 3Ј-phosphate rather than a 3Ј-hydroxyl. A 3Ј-phosphate could then be removed by polynucleotide kina...

Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5 -DNA termini and dephosphorylation of 3 -DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The smallinterfering RNA suppressed hPNK gene expression by at least 80 -90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including ␥-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiationinduced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.

Cancer stem cell studies may improve understanding of tumor pathophysiology and identify more effective strategies for cancer treatment. In a variety of organisms, Piwil2 has been implicated in multiple roles including stem cell self-renewal, RNA silencing, and translational control. In this study, we documented specific expression of the stem cell protein Piwil2 in breast cancer with predominant expression in breast cancer stem cells. In patients who were evaluated, we determined that 90% of invasive carcinomas and 81% of carcinomas in situ exhibited highest expression of Piwil2. In breast cancer cells, Piwil2 silencing suppressed the expression of signal transducer and activator of transcription 3, a pivotal regulator of Bcl-X L and cyclin D1, whose downregulation paralleled a reduction in cell proliferation and survival. Our findings define Piwil2 and its effector signaling pathways as key factors in the proliferation and survival of breast cancer stem cells. Cancer Res; 70(11); 4569-79. ©2010 AACR.