Browsing by Subject "Protein-DNA interaction"

Now showing 1 - 3 of 3
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    Building better polymerases: Engineering the replication of expanded genetic alphabets
    (Elsevier, 2020-12-11) Ouaray, Zahra; Benner, Steven A.; Georgiadis, Millie M.; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
    DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.
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    Developing small molecule inhibitors targeting Replication Protein A for platinum-based combination therapy
    (2014) Mishra, Akaash K.; Turchi, John J.; Kelley, Mark Richard, 1957-; Hurley, Thomas D., 1961-; Zhang, Zhong-Yin
    All platinum (Pt)-based chemotherapeutics exert their efficacy primarily via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of Pt-DNA lesions by nucleotide excision repair and homologous recombination (HR) can substantially reduce the effectiveness of the Pt therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions requires RPA binding to single-stranded DNA (ssDNA). We synthesized structural analogs of our previously reported RPA inhibitor TDRL-505, determined the structure activity relationships and evaluated their efficacy in tissue culture models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro and cellular activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.
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    Intrinsically disordered regions of p53 family are highly diversified in evolution
    (Elsevier, 2013) Xue, Bin; Brown, Celeste J.; Dunker, A. Keith; Uversky, Vladimir N.; Biochemistry and Molecular Biology, School of Medicine
    Proteins of the p53 family are expressed in vertebrates and in some invertebrate species. The main function of these proteins is to control and regulate cell cycle in response to various cellular signals, and therefore to control the organism's development. The regulatory functions of the p53 family members originate mostly from their highly-conserved and well-structured DNA-binding domains. Many human diseases (including various types of cancer) are related to the missense mutations within this domain. The ordered DNA-binding domains of the p53 family members are surrounded by functionally important intrinsically disordered regions. In this study, substitution rates and propensities in different regions of p53 were analyzed. The analyses revealed that the ordered DNA-binding domain is conserved, whereas disordered regions are characterized by high sequence diversity. This diversity was reflected both in the number of substitutions and in the types of substitutions to which each amino acid was prone. These results support the existence of a positive correlation between protein intrinsic disorder and sequence divergence during the evolutionary process. This higher sequence divergence provides strong support for the existence of disordered regions in p53 in vivo for if they were structured, they would evolve at similar rates as the rest of the protein.