Ribonucleotide reductase (RNR), which consists of R1 and R2 subunits, catalyzes a key step of deoxyribonucleoside triphosphate (dNTP) synthesis for DNA replication and repair. The R2 subunit is controlled in a cell cycle-specific manner for timely DNA synthesis and is negatively regulated by p53 in response to DNA damage. Herein we demonstrate that the presence of excess R2 subunits in p53(−/−) HCT-116 human colon cancer cells protects against DNA damage and replication stress. siRNA-mediated stable knockdown (>80%) of excess R2 subunits has no effect on proliferative growth but results in enhanced accumulation of γ-H2Ax and delayed recovery from DNA lesions inflicted by exposure to cisplatin and Triapine. This accentuated induction of γ-H2Ax in R2-knockdown cells is attributed to reduced ability to repair damaged DNA and overcome replication blockage. The lack of excess R2 subunits consequently augments chk1 activation and cdc25A degradation, causing impeded cell progression through the S phase and enhanced apoptosis in response to DNA damage and replication stress. In contrast, the level of R1 subunits appears to be limiting, since depletion of the R1 subunit directly activates the S phase checkpoint due to replication stress associated with impaired RNR activity. These findings suggest that excess R2 subunits facilitate DNA damage repair and recovery from replication stress through coordination with the S phase checkpoint in the absence of functional p53. Thus, the level of the R2 subunit constitutes an important determinant of the chemosensitivity of cancer cells and serves as a potential target for enhancement of DNA-damage based therapy.