Ionizing radiation (IR) is the predominant form of therapy for a wide variety of cancers. IR induces numerous variations of genomic lesions which can reduce cell proliferation and growth. The most cytotoxic of these DNA lesions is the double strand break (DSB). A DSB occurs when single stranded breaks (SSBs) occur in close proximity to one another on opposite DNA strands. IR-induced DNA strand breaks frequently contain non-ligatable 5’-hydroxyl and/or 3’- phosphate groups. Human polynucleotide kinase/phosphatase (PNKP) exhibits 5’ DNA kinase and 3’ DNA phosphatase activities, making it ideal for converting “dirty” DSB ends to compatible ends prior to ligation. This work examines DSBR kinetics of PNKP-deficient cells in an isogenic background, and demonstrates that PNKP deficient cells exhibit a late DSB repair defect in a cell cycle dependent manner. Live cell imaging demonstrates that PNKP requires its FHA domain, DNA-PKcs, and PARP for recruitment/retention to DNA damage sites in vivo.