Biochemical Activity of 17 Cancer-Associated Variants of DNA Polymerase Kappa Predicted by Electrostatic Properties.

DNA damage and repair have been widely studied in relation to cancer and therapeutics. Y-family DNA polymerases can bypass DNA lesions, which may result from external or internal DNA damaging agents, including some chemotherapy agents. Overexpression of the Y-family polymerase human pol kappa can result in tumorigenesis and drug resistance in cancer. This report describes the use of computational tools to predict the effects of single nucleotide polymorphism variants on pol kappa activity. Partial Order Optimum Likelihood (POOL), a machine learning method that uses input features from Theoretical Microscopic Titration Curve Shapes (THEMATICS), was used to identify amino acid residues most likely involved in catalytic activity. The µ4 value, a metric obtained from POOL and THEMATICS that serves as a measure of the degree of coupling between one ionizable amino acid and its neighbors, was then used to identify which protein mutations are likely to impact the biochemical activity. Bioinformatic tools SIFT, PolyPhen-2, and FATHMM predicted most of these variants to be deleterious to function. Along with computational and bioinformatic predictions, we characterized the catalytic activity and stability of 17 cancer-associated DNA pol kappa variants. We identified pol kappa variants R48I, H105Y, G147D, G154E, V177L, R298C, E362V, and R470C as having lower activity relative to wild-type pol kappa; the pol kappa variants T102A, H142Y, R175Q, E210K, Y221C, N330D, N338S, K353T, and L383F were identified as being similar in catalytic efficiency to WT pol kappa. We observed that POOL predictions can be used to predict which variants have decreased activity. Predictions from bioinformatic tools like SIFT, PolyPhen-2, and FATHMM are based on sequence comparisons and therefore are complementary to POOL but are less capable of predicting biochemical activity. These bioinformatic and computational tools can be used to identify SNP variants with deleterious effects and altered biochemical activity from a large data set.

Adapting Undergraduate Research to Remote Work to Increase Engagement.

Demand for undergraduate research experiences typically outstrips the available laboratory positions, which could have been exacerbated during the remote work conditions imposed by the SARS-CoV-2/COVID-19 pandemic. This report presents a collection of examples of how undergraduates have been engaged in research under pandemic work restrictions. Examples include a range of projects related to fluid dynamics, cancer biology, nanomedicine, circadian clocks, metabolic disease, catalysis, and environmental remediation. Adaptations were made that included partnerships between remote and in-person research students and students taking on more data analysis and literature surveys, as well as data mining, computational, and informatics projects. In many cases, these projects engaged students who otherwise would have worked in traditional bench research, as some previously had. Several examples of beneficial experiences are reported, such as the additional time spent studying the literature, which gave students a heightened sense of project ownership, and more opportunities to integrate feedback into writing and research. Additionally, the more intentional and regular communication necessitated by remote work proved beneficial for all team members. Finally, online seminars and conferences have made participation possible for many more students, especially those at predominantly undergraduate institutions. Participants aim to adopt these beneficial practices in our research groups even after pandemic restrictions end.

Mammalian DNA Polymerase Kappa Activity and Specificity.

DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

Site-Specific Reversible Protein and Peptide Modification: Transglutaminase-Catalyzed Glutamine Conjugation and Bioorthogonal Light-Mediated Removal.

Dynamic photoswitches in proteins that impart spatial and temporal control are important to manipulate and study biotic and abiotic processes. Nonetheless, approaches to install these switches into proteins site-specifically are limited. Herein we describe a novel site-specific method to generate photoremovable protein conjugates. Amine-containing chromophores (e.g., venerable  o-nitrobenzyl and less-explored o-nitrophenylethyl groups) were incorporated via transamidation into a glutamine side chain of α-gliadin, LCMV, and TAT peptides, as well as ß-casein and UmuD proteins by transglutaminase (TGase, EC 2.3.2.13). Subsequently, photolysis regenerated the native peptides and proteins. When this modification leads to the reduction or abolishment of certain activities, the process is referred to as caging, as in the case for E. coli polymerase manager protein UmuD. Importantly, this method is simple, robust, and easily adaptable, e.g., all components are commercially available.

The response of Escherichia coli to the alkylating agents chloroacetaldehyde and styrene oxide.

DNA damage is ubiquitous and can arise from endogenous or exogenous sources. DNA-damaging alkylating agents are present in environmental toxicants as well as in cancer chemotherapy drugs and are a constant threat, which can lead to mutations or cell death. All organisms have multiple DNA repair and DNA damage tolerance pathways to resist the potentially negative effects of exposure to alkylating agents. In bacteria, many of the genes in these pathways are regulated as part of the SOS reponse or the adaptive response. In this work, we probed the cellular responses to the alkylating agents chloroacetaldehyde (CAA), which is a metabolite of 1,2-dichloroethane used to produce polyvinyl chloride, and styrene oxide (SO), a major metabolite of styrene used in the production of polystyrene and other polymers. Vinyl chloride and styrene are produced on an industrial scale of billions of kilograms annually and thus have a high potential for environmental exposure. To identify stress response genes in E. coli that are responsible for tolerance to the reactive metabolites CAA and SO, we used libraries of transcriptional reporters and gene deletion strains. In response to both alkylating agents, genes associated with several different stress pathways were upregulated, including protein, membrane, and oxidative stress, as well as DNA damage. E. coli strains lacking genes involved in base excision repair and nucleotide excision repair were sensitive to SO, whereas strains lacking recA and the SOS gene ybfE were sensitive to both alkylating agents tested. This work indicates the varied systems involved in cellular responses to alkylating agents, and highlights the specific DNA repair genes involved in the responses.

Characterization of Nine Cancer-Associated Variants in Human DNA Polymerase κ.

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol κ are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol κ whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol κ variants generally fall into three categories: similar activity to wild-type (WT) pol κ (L21F, I39T, P169T, F192C, and E292K), more active than WT pol κ (S423R), and less active than pol κ (R219I, R298H, and Y432S). Of these, only pol κ variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol κ with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol κ with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol κ adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD'2C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

Prediction of distal residue participation in enzyme catalysis.

A scoring method for the prediction of catalytically important residues in enzyme structures is presented and used to examine the participation of distal residues in enzyme catalysis. Scores are based on the Partial Order Optimum Likelihood (POOL) machine learning method, using computed electrostatic properties, surface geometric features, and information obtained from the phylogenetic tree as input features. Predictions of distal residue participation in catalysis are compared with experimental kinetics data from the literature on variants of the featured enzymes; some additional kinetics measurements are reported for variants of Pseudomonas putida nitrile hydratase (ppNH) and for Escherichia coli alkaline phosphatase (AP). The multilayer active sites of P. putida nitrile hydratase and of human phosphoglucose isomerase are predicted by the POOL log ZP scores, as is the single-layer active site of P. putida ketosteroid isomerase. The log ZP score cutoff utilized here results in over-prediction of distal residue involvement in E. coli alkaline phosphatase. While fewer experimental data points are available for P. putida mandelate racemase and for human carbonic anhydrase II, the POOL log ZP scores properly predict the previously reported participation of distal residues.

Cryptocaryol Structure-Activity Relationship Study of Cancer Cell Cytotoxicity and Ability to Stabilize PDCD4.

The synthetic cryptocaryols A and B and a series of their analogues have been evaluated for their cytotoxicity and their ability to stabilize the tumor suppressor PDCD4. Cytotoxicities in the 3 to 30 µM range were found. Both the cytotoxicity and PDCD4 stabilizing ability were tolerant of large stereochemical changes to the molecule. Co-dosing studies with cryptocaryols A and B and several known cancer drugs showed no measuable enhancement in cancer drug cytotoxicity.

Structure activity relationship study of Mezzettiasides natural products and their four new disaccharide analogues for anticancer/antibacterial activity.

Ten members of the mezzettiaside family of natural products were synthesized and evaluated for anticancer and antibacterial activity. Complete anticancer (H460) and antibacterial (B. subtilis) activities for the ten natural products and four new analogues were found. Comparison to the cleistrioside and cleistetroside classes of natural products were made.

Structure Activity Relationship Study of the Cleistriosides and Cleistetrosides for Antibacterial/Anticancer Activity.

Two known cleistriosides and six known cleistetrosides were synthesized and evaluated for anticancer and antibacterial activity. This study, for the first time, reports anticancer activity and comprehensively the antibacterial activity for these oligosaccharide natural products. In addition, two new unnatural cleistetroside analogues were synthesized and tested. Biological activities for the ten oligosaccharides against B. subtilis were found to range between 4 and >64 µM, and for NCI-H460 human lung cancer epithelial cells between 7.5 and 90.9 µM. Similar activities were found for seven of the oligosaccharides against the NCI panel of 60 cell lines. The degree of acylation and location of the specific acetate groups had significant effects on the anticancer and antibacterial activity of both the cleistriosides and cleistetrosides.

A tale of two isomerases: compact versus extended active sites in ketosteroid isomerase and phosphoglucose isomerase.

Understanding the catalytic efficiency and specificity of enzymes is a fundamental question of major practical and conceptual importance in biochemistry. Although progress in biochemical and structural studies has enriched our knowledge of enzymes, the role in enzyme catalysis of residues that are not nearest neighbors of the reacting substrate molecule is largely unexplored experimentally. Here computational active site predictors, THEMATICS and POOL, were employed to identify functionally important residues that are not in direct contact with the reacting substrate molecule. These predictions then guided experiments to explore the active sites of two isomerases, Pseudomonas putida ketosteroid isomerase (KSI) and human phosphoglucose isomerase (PGI), as prototypes for very different types of predicted active sites. Both KSI and PGI are members of EC 5.3 and catalyze similar reactions, but they represent significantly different degrees of remote residue participation, as predicted by THEMATICS and POOL. For KSI, a compact active site of mostly first-shell residues is predicted, but for PGI, an extended active site in which residues in the first, second, and third layers around the reacting substrate are predicted. Predicted residues that have not been previously tested experimentally were investigated by site-directed mutagenesis and kinetic analysis. In human PGI, single-point mutations of the predicted second- and third-shell residues K362, H100, E495, D511, H396, and Q388 show significant decreases in catalytic activity relative to that of the wild type. The results of these experiments demonstrate that, as predicted, remote residues are very important in PGI catalysis but make only small contributions to catalysis in KSI.

Crystal structure of a metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis: Computational prediction and experimental validation of phosphoesterase activity.

The crystal structures of an unliganded and adenosine 5'-monophosphate (AMP) bound, metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a polymerase and histidinol phosphatase (PHP, Pfam: PF02811) domain with a second domain (residues 105-178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain are unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. Theoretical microscopic anomalous titration curve shapes, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA-proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated.

Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein.

We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single lambda-DNA molecules through the helix-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7's capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10, 25, and 100 nM concentrations respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC's nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity.

Amino acid activation of a dual-specificity tRNA synthetase is independent of tRNA.

Transfer RNA can play a role in amino acid activation by aminoacyl-tRNA synthetases. For the prolyl-tRNA synthetase (ProRS) of Methanococcus jannaschii, which activates both proline and cysteine, the role of tRNA in amino acid selection and activation is of interest in the effort to understand the mechanism of the dual-specificity. While activation of proline does not require tRNA, whether or not tRNA is required in the activation of cysteine has been a matter of debate. Here, investigation of a series of buffer conditions shows that activation of cysteine occurs without tRNA in a wide-range of buffers. However, the extent of cysteine activation is strongly buffer-dependent, varying over a 180-fold range. In contrast, the extent of proline activation is much less sensitive to buffer conditions, varying over only a 36-fold range. We also find that addition of tRNA has a small threefold stimulatory effect on cysteine activation. The lack of a major role of tRNA in activation of cysteine suggests that the dual-specificity enzyme must distinguish cysteine from proline directly, without the assistance of each cognate tRNA, to achieve the necessary specificity required for protein synthesis.