Persistent 3′-phosphate termini and increased cytotoxicity of radiomimetic DNA double-strand breaks in cells lacking polynucleotide kinase/phosphatase despite presence of an alternative 3′-phosphatase

• Published in: DNA repair Vol. 68; pp. 12 - 24
• Main Authors: Chalasani, Sri Lakshmi, Kawale, Ajinkya S., Akopiants, Konstantin, Yu, Yaping, Fanta, Mesfin, Weinfeld, Michael, Povirk, Lawrence F.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2018
• Subjects: DNA - drug effects; DNA - metabolism; DNA - radiation effects; DNA Breaks, Double-Stranded; DNA end processing; DNA End-Joining Repair; DNA Repair Enzymes - genetics; DNA Repair Enzymes - metabolism; Gene Knockdown Techniques; HCT116 Cells; HeLa Cells; Humans; Neocarzinostatin; Nonhomologous end joining; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; PNKP; Radiation, Ionizing; Radiosensitization; Zinostatin - toxicity; Nonhomologous end joining; Radiosensitization; DNA end processing; PNKP; Neocarzinostatin
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2018.05.002

Polynucleotide kinase/phosphatase (PNKP) has been implicated in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). To assess the consequences of PNKP deficiency for NHEJ of 3′-phosphate-ended DSBs, PNKP-deficient derivatives of HCT116 and of HeLa cells were generated using CRISPR/CAS9. For both cell lines, PNKP deficiency conferred sensitivity to ionizing radiation as well as to neocarzinostatin (NCS), which specifically induces DSBs bearing protruding 3′-phosphate termini. Moreover, NCS-induced DSBs, detected as 53BP1 foci, were more persistent in PNKP −/− HCT116 cells compared to their wild-type (WT) counterparts. Surprisingly, PNKP-deficient whole-cell and nuclear extracts were biochemically competent in removing both protruding and recessed 3′-phosphates from synthetic DSB substrates, albeit much less efficiently than WT extracts, suggesting an alternative 3′-phosphatase. Measurements by ligation-mediated PCR showed that PNKP-deficient HeLa cells contained significantly more 3′-phosphate-terminated and fewer 3′-hydroxyl-terminated DSBs than parental cells 5–15 min after NCS treatment, but this difference disappeared by 1 h. These results suggest that, despite presence of an alternative 3′-phosphatase, loss of PNKP significantly sensitizes cells to 3′-phosphate-terminated DSBs, due to a 3′-dephosphorylation defect.