| Among three major replicative DNA polymerases of the B-family, Pol α, Pol δ and Pol ε, Pol δ plays an essential role in chromosomal DNA replication and is also involved in various DNA repair processes in eukaryotes. Human Pol δ is commonly viewed as a heterotetrameric complex, consisting of the catalytic subunit p125 and second subunit p50, together with two additional accessory subunits, p68 and p12. The p125 is tightly associated with p50 subunit, forming a core enzyme. A growing body of research has shown that the accessory subunits play a critical role in the regulation of Pol δ functions. Mammalian cells have active defense mechanisms to deal with DNA damage, known as DN A damage response(DDR). It involves the recruitment and assembly of large complexes of proteins that orchestrate and prioritize a network of responses that includes DNA repair, activation of cell cycle checkpoints, and the decision for apoptosis.Pol δ3 is found by virtue of the depletion of p12 through the ubiquitin–proteasome pathway in response to DNA damages that are trigged by UV irradiation, alkylating agents, oxidative and replication stresses. Pol δ3 exhibits significant differences in properties to its progenitor with a major impact on cellular processes in genomic surveillance, DNA replication, and DNA repair. The p12 degradation seems to be regulated by multiple E3 ligases in response to genotoxic agents that trigger different DNA damage signaling pathways. But so far, only two E3 ligases, RNF8 and CRL4 Cdt 2, have been identified to be involved in targeting of p12 for its proteasomal degradation in response to UV triggered DNA damage. Previous sdudies show that p68 subunit could also be modified by ubiquitination and sumoylation(small ubiquitin- like modification) in vivo. However, the questions of, upon DNA damage, how many and which subunit(s) of Pol δ are modified by ubiquitination, it is poly-ubiquitinated or mono- ubiquitinated, and which E2/E3 systems are involved, remain to be further investigated.Ubiquitination and(or) sumoylation are concerned in almost all the major processes of DDR, more or less. They are involved in a variety of DNA repair processes, such as translesion synthesis, nucleotide excision, homologous recombination, etc. Based on the important role of ubiquitination, this thesis will conduct the analysis on the modification of individual subunit(s) of Pol δ by ubiquitination to reveal its novel function in DNA damage responses.In this study, a ring finger protein 8(RNF8) which functions as E3 ligase was prepared by use of baculovirus-based expression system. By the in vitro analysis system, the ubiquitination of individual subunit(s) of Pol δ was analyzed using RNF8 as E3 ligase, combining with several commencial E2 s. Also, modification of p50 and p12 subunits by ubiquitination was analyzed by RAD6/RAD18 E2/E3 system. The results showed that the RNF8 could mediate mono- ubiquitination of p50 subunit and poly- ubiquitination of p12 subunit. While for p125 subunit, there was no any modifications were observed. Intrestingly, RAD6/RAD18 could mediate ubiquitination of p50 and p12 subunits in presence of DN A substrate, respectively.Cancer and other diseases pose a major threat to human health and survival, which is one of the most important social issues over the world. Our studies could significantly contribute to the understanding of human disease by uncovering novel mechanisms that are involved in maintaining genomic stability and contro lling cell proliferation. Our works provided a basis for designing a new diagnosis and therapy of cancer by taking Pol δ as a potential target for small molecule inhibitors. |
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