Nucleobase-Modified Nucleosides and Nucleotides: Applications in Biochemistry, Synthetic Biology, and Drug Discovery

DNA is often referred to as the molecule of life since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, recent chemical and biochemical studies using nucleobase-modified nucleotides that contain non-hydrogen bonding functional groups have challenged many of the dogmatic views for the necessity of hydrogen-bonding interactions for DNA stability and function. Based on years of exciting research, this area has expanded tremendously and is thus too expansive to provide a comprehensive review on the topic. As such, this review article provides an opinion highlighting how nucleobase-modified nucleotides are being applied in diverse biomedical fields, focusing on three exciting areas of research. The first section addresses how these analogs are used as mechanistic probes for DNA polymerase activity and fidelity during replication. This section outlines the synthetic logic and medicinal chemistry approaches used to replace hydrogen-bonding functional groups to examine the contributions of shape/size, nucleobase hydrophobicity, and pi-electron interactions. The second section extends these mechanistic studies to provide insight into how nucleobase-modified nucleosides are used in synthetic biology. One example is through expansion of the genetic code in which changing the composition of DNA makes it possible to site-specifically incorporate unnatural amino acids bearing unique functional groups into enzymes and receptors. The final section describes results of pre-clinical studies using nucleobase-modified nucleosides as potential therapeutic agents against diseases such as cancer

DNA Polymerases: Perfect Enzymes for An Imperfect World

This Special Thematic Issue explores the molecular properties of DNA polymerases as extraordinary biological catalysts. In this short introductory chapter, I briefly highlight some of the most important concepts from the articles contained within this Special Issue. The contents of this Special Issue are arranged into distinct sub-categories corresponding to mechanistic studies of faithful DNA polymerization, studies of specialized DNA polymerases that function on damaged DNA, and DNA polymerases that are of therapeutic importance against various diseases. Emphasis is placed on understanding the dynamic cellular roles and biochemical functions of DNA polymerases, and how their structure and mechanism impact their cellular roles

Non-Natural Nucleotides As Probes For The Mechanism And Fidelity Of DNA Polymerases

DNA is a remarkable macromolecule that functions primarily as the carrier of the genetic information of organisms ranging from viruses to bacteria to eukaryotes. The ability of DNA polymerases to efficiently and accurately replicate genetic material represents one of the most fundamental yet complex biological processes found in nature. The central dogma of DNA polymerization is that the efficiency and fidelity of this biological process is dependent upon proper hydrogen-bonding interactions between an incoming nucleotide and its templating partner. However, the foundation of this dogma has been recently challenged by the demonstration that DNA polymerases can effectively and, in some cases, selectively incorporate non-natural nucleotides lacking classic hydrogen-bonding capabilities into DNA. In this review, we describe the results of several laboratories that have employed a variety of non-natural nucleotide analogs to decipher the molecular mechanism of DNA polymerization. The use of various non-natural nucleotides has lead to the development of several different models that can explain how efficient DNA synthesis can occur in the absence of hydrogen-bonding interactions. These models include the influence of steric fit and shape complementarity, hydrophobicity and solvation energies, base-stacking capabilities, and negative selection as alternatives to rules invoking simple recognition of hydrogen-bonding patterns. Discussions are also provided regarding how the kinetics of primer extension and exonuclease proofreading activities associated with high-fidelity DNA polymerases are influenced by the absence of hydrogen-bonding functional groups exhibited by non-natural nucleotides

Exploring New Chemotherapeutic Strategies Against Brain Cancer

Approximately 4,000 children in the United States are diagnosed each year with a brain tumor. Brain cancers are the deadliest of all pediatric cancers as they have survival rates of less than 20%. Typical treatments include surgery and radiation therapy. However, chemotherapy is the primary therapeutic option for children, especially against aggressive brain tumors. An important chemotherapeutic agent is temozolomide, an alkylating agent that causes cell death by damaging DNA. In this project, we tested the ability of non-natural nucleosides developed in our lab in order to increase the ability of temozolomide to kill brain cancer cells. Our results show that combining low doses of our nucleoside with temozolomide kills more cells compared to treatment with either compound individually. The increase in efficacy is specific for temozolomide as similar effects are not observed in cells treated with other chemotherapeutic agents such as cisplatin, 5-fluorouracil, and taxol. High-field microscopy techniques demonstrate that the combination of our nucleoside and temozolomide causes cell death via apoptosis as opposed to necrosis. A model is provided describing how our novel nucleoside analog increases the cell-killing effects of temozolomide by inhibiting the misreplication of damaged DNA created by this agent. Collectively, these studies provide pharmacological evidence for a new treatment strategy to more effectively treat patients with brain cancers.https://engagedscholarship.csuohio.edu/u_poster_2013/1007/thumbnail.jp

The Use of Non-natural Nucleotides to Probe Template-Independent DNA Synthesis

The vast majority of DNA polymerases use the complementary templating strand of DNA to guide each nucleotide incorporation. There are instances, however, in which polymerases can efficiently incorporate nucleotides in the absence of templating information. This process, known as translesion DNA synthesis, can alter the proper genetic code of an organism. To further elucidate the mechanism of template-independent DNA synthesis, we monitored the incorporation of various nucleotides at the “blunt-end” of duplex DNA by the high-fidelity bacteriophage T4 DNA polymerase. Although natural nucleotides are not incorporated at the blunt-end, a limited subset of non-natural indolyl analogues containing extensive π-electron surface areas are efficiently utilized by the T4 DNA polymerase. These analogues possess high binding affinities that are remarkably similar to those measured during incorporation opposite an abasic site. In contrast, the kpol values are significantly lower during blunt-end extension when compared to incorporation opposite an abasic site. These kinetic differences suggest that the single-stranded region of the DNA template plays an important role during polymerization through stacking interactions with downstream bases, interactions with key amino acid residues, or both. In addition, we demonstrate that terminal deoxynucleotide transferase, a template-independent enzyme, can efficiently incorporate many of these non-natural nucleotides. However, that this unique polymerase cannot extend large, bulky non-natural nucleotides suggests that elongation is limited by steric constraints imposed by structural features present within the polymerase. Regardless, the kinetic data obtained from using either DNA polymerase indicate that template-independent synthesis can occur without the contributions of hydrogen-bonding interactions and suggest that π-electron interactions play an important role in polymerization efficiency when templating information is not present

Adenosine Triphosphate-Dependent Degradation of A Fluorescent λ N Substrate Mimic by Lon Protease

Escherichia coli Lon exhibits a varying degree of energy requirement toward hydrolysis of different substrates. Efficient degradation of protein substrates requires the binding and hydrolysis of ATP such that the intrinsic ATPase of Lon is enhanced during protein degradation. Degradation of synthetic tetrapeptides, by contrast, is achieved solely by ATP binding with concomitant inhibition of the ATPase activity. In this study, a synthetic peptide (FRETN 89-98), containing residues 89–98 of λ N protein and a fluorescence donor (anthranilamide) and quencher (3-nitrotyrosine), has been examined for ATP-dependent degradation by E. coli and human Lon proteases. The cleavage profile of FRETN 89-98 by E. coli Lon resembles that of λ N degradation. Both the peptide and protein substrates are specifically cleaved between Cys93 and Ser94 with concomitant stimulation of Lon\u27s ATPase activity. Furthermore, the degradation of FRETN 89-98 is supported by ATP and AMPPNP but not ATPγS nor AMPPCP. FRETN 89-98 hydrolysis is eight times more efficient in the presence of 0.5 mM ATP compared to 0.5 mM AMPPNP at 86 μM peptide. The ATP-dependent hydrolysis of FRETN 89-98 displays sigmodial kinetics. The kcat, [S]0.5, and the Hill coefficient of FRETN 89-98 degradation are 3.2 ± 0.3 s−1, 106 ± 21 μM, and 1.6 respectively

Fluorescent Analysis of Translesion DNA Synthesis by Using A Novel, Non-natural Nucleotide Analogue

The replication of damaged DNA is a promutagenic process that can lead to disease development. This report evaluates the dynamics of nucleotide incorporation opposite an abasic site, a commonly formed DNA lesion, by using two fluorescent nucleotide analogues, 2-aminopurine deoxyribose triphosphate (2-APTP) and 5-phenylindole deoxyribose triphosphate (5-PhITP). In both cases, the kinetics of incorporation were compared by using a 32 P-radiolabel extension assay versus a fluorescence-quenching assay. Although 2-APTP is efficiently incorporated opposite a templating nucleobase (thymine), the kinetics for incorporation opposite an abasic site are significantly slower. The lower catalytic efficiency hinders its use as a probe to study translesion DNA synthesis. In contrast, the rate constant for the incorporation of 5-PhITP opposite the DNA lesion is 100-fold faster than that for 2- APTP. Nearly identical kinetic parameters are obtained from fluorescence quenching or the 32 P-radiolabel assay. Surprisingly, distinct differences in the kinetics of 5-PhITP incorporation opposite the DNA lesion are detected when using either bacteriophage T4 DNA polymerase or the Escherichia coli Klenow fragment. These differences suggest that the dynamics of nucleotide incorporation opposite an abasic site are polymerase-dependent. Collectively, these data indicate that 5-PhITP can be used to perform real time analyses of translesion DNA synthesis as well as to functionally probe differences in polymerase function

Terminal Deoxynucleotidyl Transferase: The Story of A Misguided DNA Polymerase

Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis

Evaluating The Effects of Enhanced Processivity and Metal Ions on Translesion DNA Replication Catalyzed by The Bacteriophage T4 DNA Polymerase

The fidelity of DNA replication is achieved in a multiplicative process encompassing nucleobase selection and insertion, removal of misinserted nucleotides by exonuclease activity, and enzyme dissociation from primer/templates that are misaligned due to mispairing. In this study, we have evaluated the effect of altering these kinetic processes on the dynamics of translesion DNA replication using the bacteriophage T4 replication apparatus as a model system. The effect of enhancing the processivity of the T4 DNA polymerase, gp43, on translesion DNA replication was evaluated using a defined in vitro assay system. While the T4 replicase (gp43 in complex with gp45) can perform efficient, processive replication using unmodified DNA, the T4 replicase cannot extend beyond an abasic site. This indicates that enhancing the processivity of gp43 does not increase unambiguously its ability to perform translesion DNA replication. Surprisingly, the replicase composed of an exonuclease-deficient mutant of gp43 was unable to extend beyond the abasic DNA lesion, thus indicating that molecular processes involved in DNA polymerization activity play the predominant role in preventing extension beyond the non-coding DNA lesion. Although neither T4 replicase complex could extend beyond the lesion, there were measurable differences in the stability of each complex at the DNA lesion. Specifically, the exonuclease-deficient replicase dissociates at a rate constant, koff, of 1.1 s−1 while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (koff 0.009 s−1). The increased lifetime of the wild-type replicase suggests that idle turnover, the partitioning of the replicase from its polymerase to its exonuclease active site, may play an important role in maintaining fidelity. Further attempts to perturb the fidelity of the T4 replicase by substituting Mn2+ for Mg2+ did not significantly enhance DNA synthesis beyond the abasic DNA lesion. The results of these studies are interpreted with respect to current structural information of gp43 alone and complexed with gp45

The 2′-Phosphate of NADP Is Critical for Optimum Productive Binding to 6-Phosphogluconate Dehydrogenase from Candida Utilis

Initial velocity studies obtained with alternative dinucleotide substrates for the 6-phosphogluconate dehydrogenase reaction suggest that the 2′-phosphate is critical for the optimum productive binding of the dinucleotide substrate. Initial velocity patterns obtained by varying 6-phosphogluconate at different fixed levels of NAD are nearly parallel with apparent competitive substrate inhibition by 6-phosphogluconate at pH 7 and below but intersect to the left of the ordinate at pH 8 and above. Dead-end inhibition studies indicate that the mechanism is random at all pH values. Data are interpreted in terms of a random mechanism with marked antagonism in the binding of NAD and 6-phosphogluconate at low pH. Deuterium isotope effects on V and V/K for either substrate are equal at pH 8, indicating that the kinetic mechanism is rapid equilibrium random. A decrease in the pH and the subsequent protonation of the active site general base or some other enzyme residue with a similar pK apparently results in the ineffective binding of NAD. The latter suggests either a link between the protonation state of this group and the conformation of the dinucleotide binding site or an interaction between the two