| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 89 === RPA (replication protein A) is a ubiquitous eukaryotic single-stranded DNA binding protein complex , that is composed of RPA1, RPA2 and RPA3 subunits. The heteraotrimeric complex has a high affinity for single stranded and damage DNA , and play key roles in DNA replication , repair and recombination. RPA function is mediated by its phosphorylation and protein interactions. In the previous studies, RPA1 can interact with SV40 large T antigen , DNA polymerase α ,VP16 and p53,and RPA2 can interact with XPA, RAD52 and Uracil-DNA glycosylase which are involved in DNA repair and recombination . In the other hand, the phosphorylation of RPA2 seen at G1/S transition and counting through late M phase . Besides, RPA2 become hyperphosphorylated in response to DNA-damaging agents, such as UV or ionizing irradiation , and treatment with replication inhibitor .Recent studies indicate that some kinases (DNA-PK,ATM) phosphorylate RPA2 in the response of replication-mediated DNA damage to an S-phase checkpoint mechanism .The phosphorylation of RPA2 also prevents the association of RPA with p53. Therefore, RPA phosphorylation appears to act in cellular signaling pathway and may play an important role in DNA metabolism and the regulation of cell cycle. However, the physiological role of phosphorylation of RPA is less clearly understood. The purpose of this study to identity RPA2 binding partners to further elucidate the biochemical and functional characteristics of RPA phosphorylation . Thus, protein—protein interacts of RPA2 were screening by the yeast-two-hybrid system. The initial screening of 5×104 clones, only one contained sequences identical to the full length of 4E-BP3(eukaryotic translation initiation factor 4E-binding protein 3). We confirmed RPA2-4E-BP3 binding in mammalian two hybrid system , glutathione S-transferase(GST) and co-immunoprecipitation. These result revealed that 4E-BP3 is associated with RPA2.
4E-BP3 is a 14.6KDa protein that prevents eIF4E (eukaryotic initiation factor 4E) association with eIF4G to form the eIF4F complex , resulting in the subsequent inhibition of the 43S pre-initiation complex binding to the mRNA. The binding of 4E-BP3 to eIF4E inhibits translation initiation rate of protein synthesis. In the future, the interaction of 4E-BP3 with RPA2; especially with which form (hypo-,hyper-,or dephosphorylated) of RPA2 in the cell will be further investigate.
Klebsiella pneumoniae is the common pathogen of nosocomial and community-acquired infections, especially bacterium, pneumonia, and urinary tract infections. In Taiwan, the high incidence of K. pneumoniae pyogenic liver abscess during the period form 1980(30%) to 1990(82.1%) was never reported in English literature. The high mortality rate and high incidence of K. pneumoniae pyogenic liver abscess in Taiwan make further investigation is necessary.
The exact mechanisms of increasing incidence of K. pneumoniae liver abscess in Taiwan is unclear. Since, OMP2 (outer membrane protein 2)has been cloned for a highly virulent stream of K.p129 (with a LD50 of 1.2×101 to K. pneumoniae) in our previous study. Further investigate whether OMP2 is a essential virulent determinant for K. pneumoniae or not , protective effect of anti-OMP2 antibody in experimental K. pneumoniae infection was performed. The results showed that anti- OMP2 antibody has significantly protective effect when administered before acute lethal infection. The correlation of OMP2 with respect to the virulence of K. pneumoniae will be further examined. Furthermore, several isogenic OMP2-deficient strains were constructed by using integrational plasmid to disrupt the OMP2 gene. The deficient strains will be further examined.
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