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DNA-Binding Proteins and DNA-Synthesizing Enzymes in Eukaryotes
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
The mechanism of these proteins involving in DNA replication is not known. However, a detailed analysis of the interaction of the E. coli DNA binding protein with DNA polymerase II has suggested a model to account for these various observations. E. coli DNA binding protein interacts with E. coli DNA polymerase II to form a protein-protein complex in the absence of DNA. DNA-binding protein, when bound to DNA, retains its ability to interact with DNA polymerase II and to form a ternary complex between DNA-binding protein, DNA polymerase II, and DNA. Neither E. coli DNA polymerase I nor DNA polymerase III nor phage T4-induced DNA polymerase interacts with DNA-binding protein. Thus, it appears that the specific stimulation is due to complex formation. The stimulation rate and extent of synthesis seen when the DNA-binding protein is added can be explained by destabilizing the double helix of DNA template as well as by binding to the polymerase during DNA synthesis, such that the polymerase does not dissociate from the template during processing.
Regulation of Cell Functions
Published in Enrique Pimentel, Handbook of Growth Factors, 2017
Cloning, sequence, and expression of a yeast analog of mammalian cyclin have been reported.286,287 Yeast cyclin interacts with DNA polymerase III, an enzyme that is the yeast counterpart of mammalian DNA polymerase-δ.288 Cyclin is synthesized at relatively high levels in late G1 and early S phase of the cell cycle in Saccharomyces cerevisiae, but DNA synthesis is not required for its synthesis.
Optical Imaging Probes
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
Reconstitution or complementation (i.e., the reconstitution of functional activity rather than the integrity of protein) of a split-reporter protein (firefly and RLucs) were previously used for constructing more robust protein sensors. These fragmented reporters could be used for imaging nuclear receptor translocation into the nucleus or protein-protein interactions in the cell. Initially this was achieved by using intein-driven approach (46,47). Intein (DnaE) is a catalytic subunit of DNA polymerase III. To utilize intein-driven protein splicing, the full length of the nuclear receptor [e.g., androgen receptor (48)], split RLuc, and split DnaE (a protein splicing element) were used. The obtained reporting construct consisted of (i) cytoplasmic receptor–containing fusion protein with C-terminal fragments of DnaE and luciferase and (ii) nucleus-localized fusion counterpart containing N-terminal halves of DnaE and RLuc. These two fusion proteins exhibit no luciferase activity. Upon ligand (dihydrotestosterone) stimulation, the receptor is translocated into the cellular nucleus (46,48) where protein splicing occurs as a consequence of interaction between the splicing junctions of each DnaE fusion fragment. Later it was shown that alternative approaches could be used for RLuc complementation. Rapamycin was shown to drive interaction between FK506-binding protein (FKBP12) mTOR rapamycin-binding domain (FRB) and FKBP12, which if fused to split-RLuc fragments cause the latter to regain some of its catalytic activity (49,50). A similar strategy based on split EGFP protein was also tested for in vivo imaging of subcutaneously injected cells (50).
Anti-virulence strategies for Clostridioides difficile infection: advances and roadblocks
Published in Gut Microbes, 2020
David Stewart, Farhan Anwar, Gayatri Vedantam
Multiple novel CABs have been synthesized and, by themselves, exhibit poor/negligible antibacterial activity within the concentration ranges needed to deliver their ASO cargo. However, when these CABs were combined with an ASO targeting the dnaE gene of CD (encoding the alpha subunit of DNA polymerase III), potent bacteridical activity was achieved at <12 µg/mL, with no detectable activity on Escherichia coli, Enterococcus faecalis, Bacteroides fragilis, three key representatives of the human commensal microbiota.37