Cellular redundancy of mechanisms facilitating damage-induced degradation of the polymerase delta p12 subunit

DNA polymerase delta (Pol delta) is an essential enzyme which plays critical roles in both DNA replication and DNA repair. The enzyme has four subunits: p125 and p50 which form the catalytic core, and p68 and p12, which provide increased stability, enhanced activity, and additional sites for binding of other protein partners. Previous experiments in our laboratory discovered a novel DNA damage response in which p12, the smallest subunit of Pol delta, was rapidly degraded in response to DNA damage, thereby generating a three-subunit DNA polymerase delta enzyme (Pol delta3). Subsequently, kinetic studies were performed in order to compare the enzymatic properties of Pol delta and Pol delta, and it was determined that Pol delta3 demonstrated enhanced fidelity and proofreading abilities, and increased stalling at DNA lesions. Thus, the degradation of p12 in response to DNA damage converts the Pol delta enzyme into one which can better repair DNA lesions, perhaps indicating a potential functional reason for p12 degradation.;While p12 degradation had been characterized in response to some types of DNA damage, more in-depth characterization of the agents which promote p12 breakdown was necessary. In this study, the degradation of p12 was characterized in terms of time- and dose-response to various agents which activate different DNA damage response pathways. Specifically, oxidative stress, UV-B irradiation, neocarzinostatin, and cisplatin treatments all promoted p12 breakdown, while agents that cause double stranded breaks, such as camptothecin and etoposide, did not induce p12 degradation. The activation of several DNA damage response pathways was also characterized, as the specific pathways which facilitate p12 breakdown were unknown. Through the use of siRNA and cell lines lacking specific DNA damage kinases, the ATR DNA damage signaling pathway was determined to be required for efficient p12 degradation, whereas p12 degradation was independent of the ATM pathway. The impact of the comparatively minor p38 MAPK DNA damage pathway was also characterized, and was determined to play a role in regulation of p12 under normal conditions. Following the characterization of the damaging agents and pathways that promote p12 degradation, it was logical to then identify the E3 ligase involved with p12 degradation. The CRL4 Cdt2 complex, which ubiquitinates many proteins involved in cell cycle control, was confirmed as an E3 ligase which promotes p12 degradation. The potential for post-transcriptional regulation of p12 was also examined. Through the use of microRNA mimics and inhibitors, it was evident that microRNAs-765, -920, and -3161 also play a role in the regulation of p12 levels in human cell culture.;Thus, there are several triggers and pathways which play a role in damage- induced p12 degradation, and redundant mechanisms which regulate Pol delta p12 levels. These range from activation of major and minor DNA damage pathways which likely activate the CRL4Cdt2 E3 ligase to promote p12 degradation, to non-coding microRNAs which impact p12 stability. Characterization of DNA damaging agents which trigger p12 breakdown, as well as identification of the enzymes or microRNAs directly involved in p12 degradation provides a clearer picture of the response of p12 to DNA damage, and some insights into its normal turnover. This study provides new information about the response of p12 to DNA damage, and demonstrates the importance of this process in appropriate cellular control, as there are multiple redundancies in p12 regulation.