Cellular Responses to DNA Damage

5-氟尿嘧啶（FU）是一种用于治疗晚期结肠直肠癌和其他癌症的抗癌药物，其代谢物掺入RNA和DNA中并抑制胸苷酸合酶，导致dTTP耗尽和尿嘧啶掺入DNA中。尽管MMR缺陷与FU耐受性有关，但细胞杀伤机制仍不清楚。我们研究了细胞对氟脱氧尿苷（FdU）的反应和MMR系统的作用。 在细胞短暂暴露于低剂量FdU后，MMR在S期介导DNA损伤信号传导，并以需要MutSa、MutLa和DNA复制的方式在第一个细胞周期中触发G2/M期阻滞。细胞周期阻滞由ATR激酶介导，并导致Chk 1和SMC 1的磷酸化。MutSa在体外结合FdU：G错配，这与其作为DNA损伤传感器一致。 FdU长期治疗导致G2期不可逆阻滞，这与MMR状态无关，并导致碱基切除修复途径靶向的DNA损伤累积。因此，MMR可以作为FdU介导的DNA损伤的直接传感器，通过ATR/Chk 1途径引发细胞周期阻滞。 然而，在更高水平的损伤，其他损伤监测途径，如碱基切除修复也发挥重要作用。

5-fluorouracil (FU), an anti-cancer drug used in the treatment of advanced colorectal and other cancers, and its metabolites are incorporated into RNA and DNA and inhibit thymidylate synthase resulting in depletion of dTTP and incorporation in DNA of uracil. Although MMR deficiency has been implicated in tolerance to FU, the mechanism of cell killing remains unclear. We have examined the cellular response to fluorodeoxyuridine (FdU) and the role of the MMR system. After brief exposure of cells to low doses of FdU, MMR mediates DNA damage signaling during S-phase and triggers arrest in G2/M in the first cell cycle in a manner requiring MutSa, MutLa, and DNA replication. Cell cycle arrest is mediated by ATR kinase and results in phosphorylation of Chk1 and SMC1. MutSa binds FdU:G mispairs in vitro consistent with its being a DNA damage sensor. Prolonged treatment with FdU results in an irreversible arrest in G2 that is independent of MMR status and leads to the accumulation of DNA lesions that are targeted by the base excision repair pathway. Thus, MMR can act as a direct sensor of FdU-mediated DNA lesions eliciting cell cycle arrest via the ATR/Chk1 pathway. However, at higher levels of damage, other damage surveillance pathways such as base excision repair also play important roles.