Huang, Ying; Patel, Harsh (2020)
      phageal Cancer-Related Gene 2 (ECRG2) is a novel tumor suppressor which is frequently mutated or downregulated in multiple human cancers. Previous studies have demonstrated that ECRG2 inhibits growth of cancer cells by inducing apoptotic death. However, the molecular basis of its regulation and involvement in DNA damage response remain to be elucidated. The function of tumor suppressor p53 in cellular response to stress conditions, such as DNA damage, has been well-established. In the present study, we report for the first time, that ECRG2 is a novel pro-apoptotic transcriptional target of p53 and ECRG2 expression is induced by DNA damage in a p53-dependent manner. Moreover, we demonstrate that disruption of ECRG2 leads to reduced apoptosis and improved survival following the treatment with DNA damage-inducing anticancer agent despite p53 activation in cancer cells. Significantly, we characterized a natural variant in ECRG2promoter (rs3214447) that is found in the genomes of ~38.5% of world population and showed that ECRG2 promoter with rs3214447 variant is defective in responding to p53 and DNA damage. Thus, ECRG2 is an important executor of p53-mediated apoptosis in response to DNA damage. We also report a novel biological function of ECRG2 and demonstrate that ECRG2 interacts with and stabilizes microtubules. ECRG2 was shown to protect the microtubules against the destabilization induced by cold and nocodazole treatment. In addition, we show that ECRG2 increases acetylation of microtubules, which is associated with more stable microtubules. Importantly, we demonstrate that ECRG2 disruption give rise to increased cell proliferation by elevated activation of Akt. Taken together, our findings ascribe a novel function to ECRG2 in the regulation of microtubule dynamics and cancer cell proliferation. ECRG2-mediated tumor suppressor activities elucidated in this dissertation are clinically significant. Our database analyses reveal that cancer patients with lower ECRG2expression in their tumors had poor prognosis and reduced disease-free survival as compared to their counterparts. These observations suggest that loss of ECRG2 expression and function confers survival advantage to cancer cells. Collectively, this dissertation highlights novel aspects of ECRG2 regulation and function in cancer cell sensitivity to DNA damage-inducing anticancer therapy, microtubule dynamics and cell proliferation.
    • Role of the SMC5/6 complex in DNA replication and DNA damage repair

      Feng, Wenyi; Peng, Jie (2017)
      The structural maintenance of chromosome (SMC) proteins form the core of Cohesin, Condensin and the Smc5/6 complex, which are essential for organization and metabolism of chromosomes during the cell cycle. The Smc5/6 complex is implicated a role in non-essential homologous recombination-mediated (HR) DNA repair. Inactivating Smc5/6 (via temperature-sensitivity mutant) in the S phase, but not in the G2 phase, causes mitotic failure. Hence, we hypothesize that the Smc5/6 complex has an major role in DNA replication. We analyzed the genome-wide DNA replication temporal program in different genetic backgrounds. Mapping replication fork-associated ssDNA in WT and rad53 cells in the presence of replication stress allowed us to separate early and late replication origins. Using this method, we revealed a strain background difference of origin usage in A364a and W303. We then studied the genome-wide replication dynamics in a hypomorphic mutant, smc6-P4, using a density transfer coupled with microarray method. Overall replication dynamics of the mutant are similar to that of the WT cells with exceptions in the early S phase. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We then tested whether we can exacerbate the DNA replication stress hypersensitivity of the smc6 mutant by inhibiting ribonucleotides excision repair (RER) pathway. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant’s hypersensitivity to induced replication stress. We propose that nucleotide incision with impaired Smc5/6 complex has a more disastrous outcome than the damage itself. We found that the smc6 mutant has a significant increase in ssDNA level under replication stress but inhibiting RER pathway decreases it. This result suggests that Smc5/6 complex prevents unsupervised ssDNA formation during DNA replication as a result of damage incision repair. We reason that excess ssDNA in the Smc5/6 defective cells may subsequently engage in toxic recombination. Our study thus provides novel perspectives for the role of the Smc5/ 6 complex during DNA replication.