Abstract
DNA cross-linking agent such as Mitomycin C (MMC) prevents DNA replication and activates S phase checkpoint by forming interstrand cross-links (ICLs). Though recent studies demonstrated that ubiquitylation occurs in response to various DNA damage, the analysis of ubiquitylation at stalled replication fork remain unknown. In order to analyze cellular regulation of ubiquitylation at stalled replication fork, we detected ubiquitylated protein in MMC treated cell by immunostaining with the antibody FK2 against conjugated ubiquitin. 8 hours after MMC treatment, multiple ubiquitylated proteins foci appeared approximately 80% in normal cells. Immunostaining using an antibody against cyclin A revealed that stalled replication fork-induced ubiquitylation in S phase-dependent manner. While Fanconi anemia cell lines (FA-G, FA-D2 cells) which is hypersensitivity to MMC showed ubiquitylation at the same level as normal cells, NBS1, Mre11 or BRCA1 deficient cells did not. Furthermore, we found that ubiquitylation requires several domains of NBS1, not only FHA, BRCT and Mre11 binding domain associated with homologous recombination but also ATM binding domain and ATM phosphorylation site associated with S phase checkpoint. In addition, ATM and phosphorylation site of SMC1 were also required for ubiquitylation. These results suggest that ubiquitylation at stalled replication fork functions in S phase checkpoint as well as DNA repair.
