105 research outputs found
Y-family DNA polymerases in Escherichia coli
No full textThe observation that mutations in the Escherichia coli genes umuC+ and umuD+ abolish mutagenesis induced by UV light strongly supported the counterintuitive notion that such mutagenesis is an active rather than passive process. Genetic and biochemical studies have revealed that umuC+ and its homolog dinB+ encode novel DNA polymerases with the ability to catalyze synthesis past DNA lesions that otherwise stall replication – a process termed translesion synthesis (TLS). Similar polymerases have been identified in nearly all organisms, constituting a new enzyme superfamily. Although typically viewed as unfaithful copiers of DNA, recent studies suggest that certain TLS polymerases can perform proficient and moderately accurate bypass of particular types of DNA damage. Moreover, various cellular factors can modulate their activity and mutagenic potential.National Institutes of Health (U.S.) (grant CA021615)National Institute of Environmental Health Sciences (Grant P30 ES002109
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Computational Modeling of Cancer-Related Mutations in DNA Repair Enzymes Using Molecular Dynamics and Quantum Mechanics/Molecular Mechanics
No full textThis dissertation details the use of computational methods to understand the effect that cancer-related mutations have on proteins that complex with nucleic acids. Firstly, we perform molecular dynamics (MD) simulations of various mutations in DNA polymerase κ (pol κ). Through an experimental collaboration, we classify the mutations as more or less active than the wild type complex, depending upon the incoming nucleotide triphosphate. From these classifications we use quantum mechanics/molecular mechanics (QM/MM) to explore the reaction mechanism. Preliminary analysis points to a novel method for nucleotide addition in pol κ. Secondly, we study the ten-eleven translocation 2 (TET2) enzyme in various contexts. We find that the identities of both the substrate and complementary strands (or lack thereof) are crucial for maintaining the complex structure. Separately, we find that point mutations within the protein can affect structural features throughout the complex, only at distal sites, or only within the active site. The mutation's position within the complex alone is not indicative of its impact. Thirdly, we share a new method that combines direct coupling analysis and MD to predict potential rescue mutations using poly(ADP-ribose) polymerase 1 as a model enzyme. Fourthly, we perform MD simulations of mutations in the protection of telomeres 1 (POT1) enzyme. The investigated variants modify the POT1-ssDNA complex dynamics and protein—DNA interactions. Fifthly, we investigate the incorporation of remdesivir and other nucleotide analogue prodrugs into the protein-RNA complex of severe acute respiratory syndrome-coronavirus 2 RNA-dependent RNA polymerase. We find evidence for destabilization throughout the complex and differences in inter-subunit communication for most of the incorporation patterns studied. Finally, we share a method for determining a minimum active region for QM/MM simulations. The method is validated using 4-oxalocrotonate, TET2, and DNA polymerase λ as test cases
Synthetic Nucleotides as Probes of DNA Polymerase Specificity
No full textThe genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides
Biochemical functional predictions for protein structures of unknown or uncertain function
No full textAbstractWith the exponential growth in the determination of protein sequences and structures via genome sequencing and structural genomics efforts, there is a growing need for reliable computational methods to determine the biochemical function of these proteins. This paper reviews the efforts to address the challenge of annotating the function at the molecular level of uncharacterized proteins. While sequence- and three-dimensional-structure-based methods for protein function prediction have been reviewed previously, the recent trends in local structure-based methods have received less attention. These local structure-based methods are the primary focus of this review. Computational methods have been developed to predict the residues important for catalysis and the local spatial arrangements of these residues can be used to identify protein function. In addition, the combination of different types of methods can help obtain more information and better predictions of function for proteins of unknown function. Global initiatives, including the Enzyme Function Initiative (EFI), COMputational BRidges to EXperiments (COMBREX), and the Critical Assessment of Function Annotation (CAFA), are evaluating and testing the different approaches to predicting the function of proteins of unknown function. These initiatives and global collaborations will increase the capability and reliability of methods to predict biochemical function computationally and will add substantial value to the current volume of structural genomics data by reducing the number of absent or inaccurate functional annotations
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