Search

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., Competing Interests: Conflict of interest—S. A. B. owns intellectual property associated with AEGIS nucleobases. Some of the compounds mentioned in this article are sold by Firebird Biomolecular Sciences, LLC, which is owned by S. A. B., (© 2020 Ouaray et al.)

Obtaining semisynthetic microorganisms that exploit the information density of "hachimoji" DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the "hachimoji" P:Z nucleobase pair with a similar efficiency to that seen for Watson-Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the "hachimoji" Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet., (© 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

The DNA binding domain (DBD) of the tumor suppressor p53 is the site of several oncogenic mutations. A subset of these mutations lowers the unfolding temperature of the DBD. Unfolding leads to the exposure of a hydrophobic β-strand and nucleates aggregation which results in pathologies through loss of function and dominant negative/gain of function effects. Inspired by the hypothesis that structural changes that are associated with events initiating unfolding in DBD are likely to present opportunities for inhibition, we investigate the dynamics of the wild type (WT) and some aggregating mutants through extensive all atom explicit solvent MD simulations. Simulations reveal differential conformational sampling between the WT and the mutants of a turn region (S6-S7) that is contiguous to a known aggregation-prone region (APR). The conformational properties of the S6-S7 turn appear to be modulated by a network of interacting residues. We speculate that changes that take place in this network as a result of the mutational stress result in the events that destabilize the DBD and initiate unfolding. These perturbations also result in the emergence of a novel pocket that appears to have druggable characteristics. FDA approved drugs are computationally screened against this pocket., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most p53 mutations associated with cancer are located in its DNA binding domain (DBD). Many structures (X-ray and NMR) of this domain are available in the protein data bank (PDB) and a vast conformational heterogeneity characterizes the various free and complexed states. The major difference between the apo and the holo-complexed states appears to lie in the L1 loop. In particular, the conformations of this loop appear to depend intimately on the sequence of DNA to which it binds. This conclusion builds upon recent observations that implicate the tetramerization and the C-terminal domains (respectively TD and Cter) in DNA binding specificity. Detailed PCA analysis of the most recent collection of DBD structures from the PDB have been carried out. In contrast to recommendations that small molecules/drugs stabilize the flexible L1 loop to rescue mutant p53, our study highlights a need to retain the flexibility of the p53 DNA binding surface (DBS). It is the adaptability of this region that enables p53 to engage in the diverse interactions responsible for its functionality. Proteins 2016; 84:1443-1461. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Discovery of new therapeutics is a very challenging, expensive and time-consuming process. With the number of approved drugs declining steadily, combined with increasing costs, a rational approach is needed to facilitate, expedite and streamline the drug discovery process. In silico methods are playing key roles in the discovery of a growing number of marketed drugs. The use of computational approaches, particularly molecular dynamics, in drug design is rapidly gaining momentum and acceptance as an essential part of the toolkit for modern drug discovery. From analysing atomistic details for explaining experimentally observed phenomena, to designing drugs with increased efficacy and specificity, the insight that such simulations can provide is generating new ideas and applications that have previously been unexplored. Here we discuss physics-based simulation methodologies and applications in drug design: from locating pockets to designing novel lead compounds, from small molecules to peptides. With developments in hardware, software and theory, the improved predictive abilities of in silico efforts are becoming an essential part of efficient, economic and accurate drug development strategies.