| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 107 === Transcription factor PAX3 is involved in regulating genes expression during embryonic development. Mutation in PAX3 gene is associated with Waardenburg syndrome (WS). PAX3 WS mutants are found that occur on PD and HD domain frequently. Surprisingly, PAX3 mutants have been found as a heterozygous mutant. However, the role of PAX3 WS mutants causes the disease is still unknown. Previously, we found that PAX3 loaded onto replication regions during mid-to-late S phase, while the PAX3 HD mutants could not. Moreover, PAX3 HD mutants blocked wildtype PAX3 from loading onto the heterochromatin region. Nevertheless, the function of PAX3 PD mutants remains unknown. We hypothesize that PAX3 PD mutants are dominant negative mutants involved during replication. Here, we showed that PAX3 PD mutants lost loading onto replication foci in late S phase, but loading on replication fork and interacted with PCNA. Furthermore, the loading of wildtype PAX3 onto heterochromatin was disrupted when PAX3 PD mutants expressed. PAX3 PD mutants could interact with wildtype PAX3, suggesting PAX3 PD mutants blocked wildtype PAX3 directly from heterochromatin region. Also, PAX3 PD mutants affected the interaction between PAX3 wildtype and PARP1.On the other hands, we uncovered that PAX3 PD mutants still could be PARylated. However, SUMOylation could also modify on the PAX3 LHD domain, which is PAX3 loading domain on replication fork. Showed that SUMOylation also involved in the mechanism of PAX3 loading on replication fork. We also demonstrated that PAX3 WS mutants induced replication stress by calculating the number of micronucleus and 53BP1 nuclear bodies formation. In summary, our results demonstrated that PAX3 PD mutants directly affected wildtype PAX3 load onto replication fork with PCNA until heterochromatin replicating, thus induced replication stress. Collectively, these studies showed that WS mutants were dominant negative mutants impaired wildtype PAX3 loading on replication foci, and importance of PAX3 domain for loading on replication fork. However, the mechanism was relative with RPA1 protein and the modifiers of SUMOylation and PARylation.
|
|---|
