Disruption of DNA damage response pathways results in an accumulation of genetic mutations, gene amplifications and chromosome alterations, which are key initiating factors in cellular transformation and oncogenesis. PBF is a multifunctional proto-oncogene which is overexpressed in thyroid, pituitary and breast cancers, with roles in cell transformation, invasion and transporter regulation. Recently, we reported that targeted transgenic expression of PBF promotes genetic instability (GI) in vivo. Now, we provide evidence of a direct functional interaction between PBF and the single strand DNA repair (SSDR) enzyme Ataxia telangiectasia and RAD3-related protein (ATR). In HeLa cells, PBF significantly attenuated the ATR-mediated phosphorylation of downstream DNA repair targets Chk1 and RPA32 in response to u.v. treatment. By contrast, PBF was not involved in the double-stranded break repair (DSBR) response to ionising radiation, with the phosphorylation of the key DSBR proteins γH2AX, NBS1, SMC1 and Chk2 unaffected by PBF levels in response to 10 Gy of IR. We thus confined our studies to SSDR. PBF protein expression was stabilised by increasing doses of u.v. from 20 to 130 J/m2, and with increasing time (0, 0.5, 1, 2, 3, 4, and 6 h) following 20 J/m2 u.v. treatment. Consistent findings were apparent in TT cells. FACS analysis demonstrated that u.v.-irradiation was associated with significantly increased numbers of HeLa cells entering mitosis in cells overexpressing PBF compared to vector-only control cells (0.8 vs 1.2%, P<0.005, n=5). Given that PBF induction in response to single strand DNA damage resulted in altered ATR function, we investigated whether PBF and ATR interact in vitro. Forward and reverse co-immunoprecipitation assays confirmed specific interaction between ATR and PBF in HeLa cells, although the stringency of binding did not increase over time (0, 0.5, 1, 3, and 6 h) following u.v. induction of single strand breaks. Taken together, our data demonstrate a novel potential mechanism to explain our finding that PBF induces GI in vivo; PBF binds to the single strand DNA repair enzyme ATR, dysregulating its kinase activity and decreasing the phosphorylation of RPA and Chk1 following DNA damage. Oncogenic expression of PBF in endocrine cancers would thus confer a survival and proliferative advantage following DNA damage, facilitating neoplastic growth and tumourigenesis.