Reduced Level of Ribonucleotide Reductase R2 Subunits Increases Dependence on Homologous Recombination Repair of Cisplatin-Induced DNA Damage

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) required for replicative and repair DNA synthesis. Mammalian RNR is a heteromeric enzyme consisting primarily of R1 and R2 subunits during the S phase of the cell cycle. We have previously shown that the presence of excess R2 subunits protects p53-deficient human colon cancer cells from cisplatin-induced DNA damage and replication stress. However, the mode of DNA repair influenced by changes in the level of the R2 subunit remained to be defined. In the present study, we demonstrated that depletion of BRCA1, an important factor of homologous recombination repair (HRR), preferentially sensitized stable R2-knockdown p53(-/-) HCT116 cells to the cytotoxicity of cisplatin and γ H2AX induction. In accord with this finding, these R2-knockdown cells exhibited increased dependence on HRR, as evidenced by elevated levels of cisplatin-induced Rad51 foci and sister chromatid exchange frequency. Furthermore, stable knockdown of the R2 subunit also led to decreased cisplatin-induced gap-filling synthesis in nucleotide excision repair (NER) and a reduced dATP level in the G2/M phase of the cell cycle. These results suggest that an increased level of the R2 subunit extends the availability of dATP in the G2/M phase to promote the repair of NER-mediated single-strand gaps that are otherwise converted into double-strand breaks in the subsequent S phase. We propose that HRR becomes important for recovery from cisplatin-DNA lesions when the post-excision process of NER is restrained by reduced levels of the R2 subunit and dATP in p53-deficient cancer cells. cytotoxic effects of cisplatin. We now present evidence that excess R2 subunits function to promote NER of cisplatin-induced primary DNA lesions in the G2/M phase of the cell cycle. Thus, a reduced level of the R2 subunit predisposes p53-deficient cancer cells to increase dependency on HRR of secondary lesions incurred in the subsequent S phase transition. These findings have clinical implications in treating human malignancies using RNR inhibitors in combination with platinum-based chemotherapy. foci, slides were stained with AlexaFluor 488 conjugated anti- γ -H2AX (Cell Signaling) and mouse anti-BRCA1 (D-9) (Santa Cruz) antibodies, followed by incubation with an AlexaFluor 546 conjugated anti-mouse IgG antibody (Invitrogen). Immunofluorescence of target proteins and nuclei were viewed with a Zeiss Laser Scanning System LSM510 (Zeiss, Thornwood, NY) and confocal images were analyzed with a Zeiss LSM Image Browser. flow cytometry analyses to determine the incorporation of EdU into the G2/M cell population indicative of DNA repair synthesis. Cisplatin 0.6-, and 1.0/1.4-fold, respectively, in both vector and R2-knockdown cells. Among these changes in dNTP levels, R2-knockdown cells displayed a significant reduction in the dATP level compared to vector cells in the G2/M phase. Given that the large elevation in the dATP level corresponds to replicative DNA synthesis occurring in the S phase, these results suggest that stable knockdown of the R2 subunit leads to a suppressed dATP level in the G2/M phase and negatively impacts repair DNA synthesis in p53-deficient cells.