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Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment., Competing Interests: The authors have declared that no competing interests exist.

Telomerase, the end-replication enzyme, is reactivated in malignant cancers to drive cellular immortality. While this distinction makes telomerase an attractive target for anti-cancer therapies, most approaches for inhibiting its activity have been clinically ineffective. As opposed to inhibiting telomerase, we use its activity to selectively promote cytotoxicity in cancer cells. We show that several nucleotide analogs, including 5-fluoro-2'-deoxyuridine (5-FdU) triphosphate, are effectively incorporated by telomerase into a telomere DNA product. Administration of 5-FdU results in an increased number of telomere-induced foci, impedes binding of telomere proteins, activates the ATR-related DNA-damage response, and promotes cell death in a telomerase-dependent manner. Collectively, our data indicate that telomerase activity can be exploited as a putative anti-cancer strategy., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Inhibiting DNA synthesis is an important therapeutic strategy that is widely used to treat a number of hyperproliferative diseases including viral infections, autoimmune disorders, and cancer. This chapter describes two major categories of therapeutic agents used to inhibit DNA synthesis. The first category includes purine and pyrmidine nucleoside analogs that directly inhibit DNA polymerase activity. The second category includes DNA damaging agents including cisplatin and chlorambucil that modify the composition and structure of the nucleic acid substrate to indirectly inhibit DNA synthesis. Special emphasis is placed on describing the molecular mechanisms of these inhibitory effects against chromosomal and mitochondrial DNA polymerases. Discussions are also provided on the mechanisms associated with resistance to these therapeutic agents. A primary focus is toward understanding the roles of specialized DNA polymerases that by-pass DNA lesions produced by DNA damaging agents. Finally, a section is provided that describes emerging areas in developing new therapeutic strategies targeting specialized DNA polymerases.

Translesion DNA synthesis (TLS) is the ability of DNA polymerases to incorporate nucleotides opposite and beyond damaged DNA. TLS activity is an important risk factor for the initiation and progression of genetic diseases such as cancer. In this study, we evaluate the ability of a high-fidelity DNA polymerase to perform TLS with 8-oxo-guanine (8-oxo-G), a highly pro-mutagenic DNA lesion formed by reactive oxygen species. Results of kinetic studies monitoring the incorporation of modified nucleotide analogs demonstrate that the binding affinity of the incoming dNTP is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for the polymerization step is regulated by hydrogen-bonding interactions made between the incoming nucleotide with 8-oxo-G. Results generated here for replicating the miscoding 8-oxo-G are compared to those published for the replication of the non-instructional abasic site. During the replication of both lesions, binding of the nucleotide substrate is controlled by energetics associated with nucleobase desolvation, whereas the rate constant for the polymerization step is influenced by the physical nature of the DNA lesion, that is, miscoding versus non-instructional. Collectively, these studies highlight the importance of nucleobase desolvation as a key physical feature that enhances the misreplication of structurally diverse DNA lesions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This report evaluates the pro-mutagenic behavior of 8-oxo-guanine (8-oxo-G) by quantifying the ability of high-fidelity and specialized DNA polymerases to incorporate natural and modified nucleotides opposite this lesion. Although high-fidelity DNA polymerases such as pol δ and the bacteriophage T4 DNA polymerase replicating 8-oxo-G in an error-prone manner, they display remarkably low efficiencies for TLS compared to normal DNA synthesis. In contrast, pol η shows a combination of high efficiency and low fidelity when replicating 8-oxo-G. These combined properties are consistent with a pro-mutagenic role for pol η when replicating this DNA lesion. Studies using modified nucleotide analogs show that pol η relies heavily on hydrogen-bonding interactions during translesion DNA synthesis. However, nucleobase modifications such as alkylation to the N2 position of guanine significantly increase error-prone synthesis catalyzed by pol η when replicating 8-oxo-G. Molecular modeling studies demonstrate the existence of a hydrophobic pocket in pol η that participates in the increased utilization of certain hydrophobic nucleotides. A model is proposed for enhanced pro-mutagenic replication catalyzed by pol η that couples efficient incorporation of damaged nucleotides opposite oxidized DNA lesions created by reactive oxygen species. The biological implications of this model toward increasing mutagenic events in lung cancer are discussed., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Nucleosides and their corresponding mono-, di-, and triphosphates play important roles in maintaining cellular homeostasis. In addition, perturbations in this homeostasis can result in dysfunctional cellular processes that cause pathological conditions such as cancer and autoimmune diseases. This review article discusses contemporary research areas applying nucleoside analogs to probe the mechanistic details underlying the complexities of nucleoside metabolism at the molecular and cellular levels. The first area describes classic and contemporary approaches used to quantify the activity of nucleoside transporters, an important class of membrane proteins that mediate the influx and efflux of nucleosides and nucleobases. A focal point of this section is describing how biophotonic nucleosides are replacing conventional assays employing radiolabeled substrates to study the mechanism of these proteins. The second section describes approaches to understand the utilization of nucleoside triphosphates by cellular DNA polymerases during DNA synthesis. Emphasis here is placed on describing how novel nucleoside analogs such as 5-ethynyl-2'-deoxyuridine are being used to quantify DNA synthesis during normal replication as well as during the replication of damaged DNA. In both sections, seminal research articles relevant to these areas are described to highlight how these novel probes are improving our understanding of these biological processes. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

Nucleoside transport is an essential process that helps maintain the hyperproliferative state of most cancer cells. As such, it represents an important target for developing diagnostic and therapeutic agents that can effectively detect and treat cancer, respectively. This report describes the development of a metal-containing nucleoside designated Ir(III)-PPY nucleoside that displays both therapeutic and diagnostic properties against the human epidermal carcinoma cell line KB3-1. The cytotoxic effects of Ir(III)-PPY nucleoside are both time- and dose-dependent. Flow cytometry analyses validate that the nucleoside analog causes apoptosis by blocking cell cycle progression at G2/M. Fluorescent microscopy studies show rapid accumulation in the cytoplasm within 4 h. However, more significant accumulation is observed in the nucleus and mitochondria after 24 h. This localization is consistent with the ability of the metal-containing nucleoside to influence cell cycle progression at G2/M. Mitochondrial depletion is also observed after longer incubations (Δt ∼48 h), and this effect may produce additional cytotoxic effects. siRNA knockdown experiments demonstrate that the nucleoside transporter, hENT1, plays a key role in the cellular entry of Ir(III)-PPY nucleoside. Collectively, these data provide evidence for the development of a metal-containing nucleoside that functions as a combined therapeutic and diagnostic agent against cancer., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Ionizing radiation (IR) is an important therapeutic modality used in approximately 50% of all cancer patients and is particularly effective against solid tumors that cannot be removed by surgery or that are refractory to standard anticancer agents. IR is often combined with other chemotherapeutic agents with the goal of sensitizing cancer cells to the cytotoxic effects of IR to produce a synergistic cell-killing effect., Areas Covered: This review article describes current and emerging therapeutic agents that are designed to increase the therapeutic efficacy of IR. This includes a discussion of how IR causes cell death by damaging nucleic acid. The involvement of various DNA repair pathways, cell-cycle-dependent kinases and apoptotic pathways is also described. This mechanistic information provides the framework to understand how combining therapeutic modalities with IR produces synergistic effects as well as to explain how emerging therapeutic strategies are being designed to inhibit or activate these pathways. Biochemical mechanisms and clinical applications of these chemical entities are discussed. Finally, brief descriptions are provided for several emerging chemical entities that show promise as potential adjunctive agents to sensitize cells to the effects of IR., Expert Opinion: Using DNA damaging agents or kinase inhibitors to potentiate the cytotoxic effects of IR has significantly improved patient outcomes. However, several advancements in instrumentation as well as new molecular targets are changing the landscape of applying IR as a therapeutic modality.

Fundamental study of enzymatic nucleoside transport suffers for lack of optical probes that can be tracked noninvasively. Nucleoside transporters are integral membrane glycoproteins that mediate the salvage of nucleosides and their passage across cell membranes. The substrate recognition site is the deoxyribose sugar, often with little distinction among nucleobases. Reported here are nucleoside analogues in which emissive, cyclometalated iridium(III) complexes are "clicked" to C-1 of deoxyribose in place of canonical nucleobases. The resulting complexes show visible luminescence at room temperature and 77 K with microsecond-length triplet lifetimes. A representative complex is crystallographically characterized. Transport and luminescence are demonstrated in cultured human carcinoma (KB3-1) cells., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nucleoside analogs are an important class of anticancer agent that historically show better efficacy against hematological cancers versus solid tumors. This report describes the development and characterization of a new class of nucleoside analog that displays anticancer effects against both hematological and adherent cancer cell lines. These new analogs lack canonical hydrogen-bonding groups yet are effective nucleotide substrates for several high-fidelity DNA polymerases. Permutations in the position of the non-hydrogen-bonding functional group greatly influence the kinetic behavior of these nucleosides. One particular analog designated 4-nitroindolyl-2'-deoxynucleoside triphosphate (4-NITP) is unique as it is incorporated opposite C and T with high catalytic efficiencies. In addition, this analog functions as a nonobligate chain terminator of DNA synthesis, since it is poorly elongated. Consistent with this mechanism, the corresponding nucleoside, 4-nitroindolyl-2'-deoxynucleoside (4-NIdR), produces antiproliferative effects against leukemia cells. 4-NIdR also produces cytostatic and cytotoxic effects against several adherent cancer cell lines, especially those that are deficient in mismatch repair and p53. Cell death in this case appears to occur via mitotic catastrophe, a specialized form of apoptosis. Mass spectroscopy experiments performed on nucleic acid isolated from cells treated with 4-NIdR validate that the non-natural nucleoside is stably incorporated into DNA. Xenograft mouse studies demonstrate that administration of 4-NIdR delays tumor growth without producing adverse side effects such as anemia and thrombocytopenia. Collectively, the results of in vitro, cell-based, and animal studies provide evidence for the development of a novel nucleoside analog that shows enhanced effectiveness against solid tumors.

This report describes the use of several isosteric non-natural nucleotides as probes to evaluate the roles of nucleobase shape, size, solvation energies, and π-electron interactions as forces influencing key kinetic steps of the DNA polymerization cycle. Results are provided using representative high- and low-fidelity DNA polymerases. Results generated with the E. coli Klenow fragment reveal that this high-fidelity polymerase utilizes hydrophobic nucleotide analogues with higher catalytic efficiencies compared to hydrophilic analogues. These data support a major role for nucleobase desolvation during nucleotide selection and insertion. In contrast, the low-fidelity HIV-1 reverse transcriptase discriminates against hydrophobic analogues and only tolerates non-natural nucleotides that are capable of hydrogen-bonding or π-stacking interactions. Surprisingly, hydrophobic analogues that function as efficient substrates for the E. coli Klenow fragment behave as noncompetitive or uncompetitive inhibitors against HIV-1 reverse transcriptase. In these cases, the mode of inhibition depends upon the absence or presence of a templating nucleobase. Molecular modeling studies suggest that these analogues bind to the active site of reverse transcriptase as well as to a nearby hydrophobic binding pocket. Collectively, the studies using these non-natural nucleotides reveal important mechanistic differences between representative high- and low-fidelity DNA polymerases during nucleotide selection and incorporation., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2'-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nucleoside transporters play important physiological roles by regulating intra- and extra-cellular concentrations of purine and pyrimidine (deoxy)nucleosides. This review describes the biological function and activity of the two major families of membrane nucleoside transporters that exist in mammalian cells. These include equilibrative nucleoside transporters that transport nucleosides in a gradient-dependent fashion and concentrative nucleoside transporters that import nucleosides against a gradient by coupling movement with sodium transport. Particular emphasis is placed on describing the roles of nucleoside transport in normal physiological processes, including inflammation, cardiovascular function and nutrient transport across the blood-brain barrier. In addition, the role of nucleoside transport in pathological conditions such as cardiovascular disease and cancer are discussed. The potential therapeutic applications of manipulating nucleoside transport activities are discussed, focusing on nucleoside analogs as anti-neoplastic agents. Finally, we discuss future directions for the development of novel chemical entities to measure nucleoside transport activity at the cellular and organismal level.

Acute lymphoblastic leukemia (ALL) is the most common type of childhood cancer, presenting with approximately 5,000 new cases each year in the United States. An interesting enzyme implicated in this disease is terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase involved in V(D)J recombination. TdT is an excellent biomarker for ALL as it is overexpressed in ~90% of ALL patients, and these higher levels correlate with a poor prognosis. These collective features make TdT an attractive target to design new selective anti-cancer agents against ALL. In this report, we evaluate the anti-leukemia activities of two non-natural nucleotides designated 5-nitroindolyl-2'-deoxynucleoside triphosphate (5-NITP) and 3-ethynyl-5-nitroindolyl-2'-deoxynucleoside triphosphate (3-Eth-5-NITP). Using purified TdT, we demonstrate that both non-natural nucleotides are efficiently utilized as TdT substrates. However, 3-Eth-5-NITP is poorly elongated, and this observation validates its activity as a chain-terminator for blunt-end DNA synthesis. Cell-based experiments validate that the corresponding non-natural nucleoside produces robust cytostatic and cytotoxic effects against leukemia cells that overexpress TdT. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore via "click" chemistry. This reaction allows the extent of nucleotide incorporation to be quantified such that the anti-cancer effects of the corresponding non-natural nucleoside can be self-assessed. The applications of this novel nucleoside are discussed, focusing on its use as a "theranostic" agent that can improve the accuracy of dosing regimens and accelerate clinical decisions regarding therapeutic intervention.

This report describes the design and application of several distinct gold-containing indoles as anticancer agents. When used individually, all gold-bearing compounds display cytostatic effects against leukemia and adherent cancer cell lines. However, two gold-bearing indoles show unique behavior by increasing the cytotoxic effects of clinically relevant levels of ionizing radiation. Quantifying the amount of DNA damage demonstrates that each gold-indole enhances apoptosis by inhibiting DNA repair. Both Au(I)-indoles were tested for inhibitory effects against various cellular targets including thioredoxin reductase, a known target of several gold compounds, and various ATP-dependent kinases. While neither compound significantly inhibits the activity of thioreoxin reductase, both showed inhibitory effects against several kinases associated with cancer initiation and progression. The inhibition of these kinases provides a possible mechanism for the ability of these Au(I)-indoles to potentiate the cytotoxic effects of ionizing radiation. Clinical applications of combining Au(I)-indoles with ionizing radiation are discussed as a new strategy to achieve chemosensitization of cancer cells.

The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

O(6)-methylguanine (O(6)-MeG) is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles of hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and nonnatural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O(6)-MeG. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase's active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O(6)-MeG observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of nonnatural nucleotides identified an analog that displays high selectivity for incorporation opposite O(6)-MeG compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.

This report examines the molecular mechanism by which high-fidelity DNA polymerases select nucleotides during the replication of an abasic site, a non-instructional DNA lesion. This was accomplished by synthesizing several unique 5-substituted indolyl 2'-deoxyribose triphosphates and defining their kinetic parameters for incorporation opposite an abasic site to interrogate the contributions of π-electron density and solvation energies. In general, the K(d, app) values for hydrophobic non-natural nucleotides are ∼10-fold lower than those measured for isosteric hydrophilic analogs. In addition, k(pol) values for nucleotides that contain less π-electron densities are slower than isosteric analogs possessing higher degrees of π-electron density. The differences in kinetic parameters were used to quantify the energetic contributions of desolvation and π-electron density on nucleotide binding and polymerization rate constant. We demonstrate that analogs lacking hydrogen-bonding capabilities act as chain terminators of translesion DNA replication while analogs with hydrogen bonding functional groups are extended when paired opposite an abasic site. Collectively, the data indicate that the efficiency of nucleotide incorporation opposite an abasic site is controlled by energies associated with nucleobase desolvation and π-electron stacking interactions whereas elongation beyond the lesion is achieved through a combination of base-stacking and hydrogen-bonding interactions.

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., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

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., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Most models accounting for the efficiency and fidelity of DNA polymerization invoke the use of either hydrogen bonding contacts or complementarity of shape and size between the formed base pair. This report evaluates these mechanisms by quantifying the ability of a high-fidelity DNA polymerase to replicate 5-nitroindole, a purine mimetic devoid of classic hydrogen bonding capabilities. 5-NITP acts as a universal nucleotide since it is incorporated opposite any of the four natural nucleobases with nearly equal efficiencies. Surprising, the polymerization reaction is not reciprocal as natural nucleotides are poorly incorporated opposite 5-nitroindole in the template strand. Incorporation opposite 5-nitroindole is more efficient using natural nucleotides containing various modifications that increase their base stacking potential. However, 5-substituted indolyl nucleotides that contain pi-electron and/or hydrophobic groups are incorporated opposite the non-natural nucleobase with the highest catalytic efficiencies. The collective data set indicate that replication of a non-natural nucleobase is driven by a combination of the hydrophobic nature and pi-electron surface area of the incoming nucleotide. In this mechanism, the overall hydrophobicity of the incoming nucleobase overcomes the lack of hydrogen bonding groups that are generally required for optimal DNA polymerization. However, the lack of hydrogen bonds between base pairs prevents primer extension. This final aspect is manifest by the appearance of unusually high pyrophosphorolysis activity by the T4 DNA polymerase that is only observed with the non-natural nucleobase in the template. These results highlight the importance of hydrogen bonding interactions during primer extension and pyrophosphorolysis.

Multidrug resistance during cancer chemotherapy is commonly acquired by overexpression of the ATP binding cassette transporter, P-glycoprotein (P-gp). As such, inhibitors that target P-gp activity represent potential therapeutic agents against this form of drug resistance. This study evaluated the ability of various non-natural nucleosides that mimic the core structure of adenosine to modulate drug resistance by inhibiting the ATPase activity to P-gp. Of several analogues tested, only one novel non-natural nucleoside, 5-cyclohexylindolyl-2'-deoxyribose (5-CHInd), behaves as a P-gp inhibitor. Although 5-CHInd is an adenosine analogue that should block the binding of ATP, the non-natural nucleoside surprisingly stimulates the ATPase activity of P-gp in vitro. However, 5-CHInd is not an exportable substrate for P-gp as it is not transported across an MDCK-MDR1 monolayer. In addition, 5-CHInd differentially modulates MDR by decreasing or increasing the cytotoxicity of several chemotherapeutic agents. Although 5-CHInd displays variable activity in modulating the efflux of various drugs by P-gp, there is a correlation between changes observed in the drug-stimulated ATPase catalytic efficiency induced by 5-CHInd and its effect on drug efflux. The paradoxical behavior of 5-CHInd is rationalized within the context of contemporary models of P-gp function. In addition, the data are used to develop a predictive in vitro model for rapidly identifying potential drug-drug interactions with P-gp.

Translesion DNA synthesis represents the ability of a DNA polymerase to incorporate and extend beyond damaged DNA. In this report, the mechanism and dynamics by which the Escherichia coli Klenow fragment performs translesion DNA synthesis during the misreplication of an abasic site were investigated using a series of natural and non-natural nucleotides. Like most other high-fidelity DNA polymerases, the Klenow fragment follows the "A-rule" of translesion DNA synthesis by preferentially incorporating dATP opposite the noninstructional lesion. However, several 5-substituted indolyl nucleotides lacking classical hydrogen-bonding groups are incorporated approximately 100-fold more efficiently than the natural nucleotide. In general, analogues that contain large substituent groups in conjunction with significant pi-electron density display the highest catalytic efficiencies ( k cat/ K m) for incorporation. While the measured K m values depend upon the size and pi-electron density of the incoming nucleotide, k cat values are surprisingly independent of both biophysical features. As expected, the efficiency by which these non-natural nucleotides are incorporated opposite templating nucleobases is significantly reduced. This reduction reflects minimal increases in K m values coupled with large decreases in k cat values. The kinetic data obtained with the Klenow fragment are compared to that of the high-fidelity bacteriophage T4 DNA polymerase and reveal distinct differences in the dynamics by which these non-natural nucleotides are incorporated opposite an abasic site. These biophysical differences argue against a unified mechanism of translesion DNA synthesis and suggest that polymerases employ different catalytic strategies during the misreplication of damaged DNA.

Numerous pathological states, including cancer, autoimmune diseases, and viral/bacterial infections, are often attributed to uncontrollable DNA replication. Inhibiting this essential biological process provides an obvious therapeutic target against these diseases. A logical target is the DNA polymerase, the enzyme responsible for catalyzing the addition of mononucleotides to a growing polymer using a DNA or RNA template as a guide for directing each incorporation event. This review provides a summary of therapeutic agents that target polymerase activity. A discussion of the biological function and mechanism of polymerases is first provided to illustrate the strategy for therapeutic intervention as well as the rational design of various nucleoside analogues that inhibit various polymerases associated with viral infections and cancer. The development of nucleoside and non-nucleoside inhibitors as antiviral agents is discussed with particular emphasis on their mechanism of action, structure-activity relationships, toxicity, and mechanism of resistance. In addition, commonly used anticancer agents are described to illustrate the similarities and differences associated with various nucleoside analogues as therapeutic agents. Finally, new therapeutic approaches that include the inhibition of selective polymerases involved in DNA repair and/or translesion DNA synthesis as anticancer agents are discussed.

Abasic sites are mutagenic DNA lesions formed as a consequence of inappropriate modifications to the functional groups present on purines and pyrimidines. In this paper we quantify the ability of the high-fidelity bacteriophage T4 DNA polymerase to incorporate various promutagenic alkylated nucleotides opposite and beyond this class of non-instructional DNA lesions. Kinetic analyses reveal that modified nucleotides such as N6-methyl-dATP and O6-methyl-dGTP are incorporated opposite an abasic site far more effectively than their unmodified counterparts. The enhanced incorporation is caused by a 10-fold increase in kpol values that correlates with an increase in hydrophobicity as well as changes in the tautomeric form of the nucleobase to resemble adenine. These biophysical features lead to enhanced base-stacking properties that also contribute toward their ability to be easily extended when paired opposite the non-instructional DNA lesion. Surprisingly, misincorporation opposite templating DNA is not enhanced by the increased base-stacking properties of most modified purines. The dichotomy in promutagenic DNA synthesis catalyzed by a high-fidelity polymerase indicates that the dynamics for misreplicating a miscoding versus a non-instructional DNA lesion are different. The collective data set is used to propose models accounting for synergistic enhancements in mutagenesis and the potential to develop treatment-related malignancies as a consequence of utilizing DNA-damaging agents as chemotherapeutic agents.

The promutagenic process known as translesion DNA synthesis reflects the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template. To study the underlying mechanism of nucleotide selection during this process, we quantified the incorporation of various non-natural nucleotide analogs opposite an abasic site, a non-templating DNA lesion. Our kinetic studies using the bacteriophage T4 DNA polymerase reveal that the pi-electron surface area of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site. A remaining question is whether the selective insertion of these non-hydrogen-bonding analogs can be achieved through optimization of shape and pi-electron density. In this report, we describe the synthesis and kinetic characterization of four novel nucleotide analogs, 5-cyanoindolyl-2'-deoxyriboside 5'-triphosphate (5-CyITP), 5-ethyleneindolyl-2'-deoxyriboside 5'-triphosphate (5-EyITP), 5-methylindolyl-2'-deoxyriboside 5'-triphosphate (5-MeITP), and 5-ethylindolyl-2'-deoxyriboside 5'-triphosphate (5-EtITP). Kinetic analyses indicate that the overall catalytic efficiencies of all four nucleotides are related to their base-stacking properties. In fact, the catalytic efficiency for nucleotide incorporation opposite an abasic site displays a parabolic trend in the overall pi-electron surface area of the non-natural nucleotide. In addition, each non-natural nucleotide is incorporated opposite templating DNA approximately 100-fold worse than opposite an abasic site. These data indicate that selectivity for incorporation opposite damaged DNA can be achieved through optimization of the base-stacking properties of the incoming nucleotide.

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 pi-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 k(pol) 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 pi-electron interactions play an important role in polymerization efficiency when templating information is not present.

UV light causes the formation of thymine dimers that can be misreplicated to induce mutagenesis and carcinogenesis. This report describes the use of a series of non-natural indolyl nucleotides in probing the ability of the high-fidelity bacteriophage T4 DNA polymerase to replicate this class of DNA lesion. Kinetic data reveal that indolyl analogues containing large pi-electron surface areas are incorporated opposite the thymine dimer almost as effectively as an abasic site, a noninstructional lesion. However, there are notable differences in the kinetic parameters for each DNA lesion that indicate distinct mechanisms for their replication. For example, the rate constants for incorporation opposite a thymine dimer are considerably slower than those measured opposite an abasic site. In addition, the magnitude of these rate constants depends equally upon contributions from pi-electron density and the overall size of the analogue. In contrast, binding of a nucleotide opposite a thymine dimer is directly correlated with the overall pi-electron surface area of the incoming dXTP. In addition to defining the kinetics of polymerization, we also provide the first reported characterization of the enzymatic removal of natural and non-natural nucleotides paired opposite a thymine dimer through exonuclease degradation or pyrophosphorolysis activity. Surprisingly, the exonuclease activity of the bacteriophage enzyme is activated by a thymine dimer but not by an abasic site. This dichotomy suggests that the polymerase can "sense" bulky lesions to partition the damaged DNA into the exonuclease domain. The data for both nucleotide incorporation and excision are used to propose models accounting for polymerase "switching" during translesion DNA synthesis.

Translesion DNA synthesis represents the ability of a DNA polymerase to misinsert a nucleotide opposite a DNA lesion. Previous kinetic studies of the bacteriophage T4 DNA polymerase using a series of non-natural nucleotides suggest that pi-electron density of the incoming nucleotide substantially contributes to the efficiency of incorporation opposite an abasic site, a nontemplating DNA lesion. However, it is surprising that these nonhydrogen-bonding analogues can also be incorporated opposite natural templating DNA with variable degrees of efficiency. In this article, we describe attempts to achieve selectivity for incorporation opposite the abasic site through optimization of pi-electron density and shape of the nucleobase. Toward this goal, we report the synthesis and enzymatic characterization of two novel nucleotide analogues, 5-napthyl-indolyl-2'-deoxyriboside triphosphate (5-NapITP) and 5-anthracene-indolyl-2'-deoxyriboside triphosphate (5-AnITP). The overall catalytic efficiency for their incorporation opposite an abasic site is similar to that of other non-natural nucleotides such as 5-NITP and 5-PhITP that contain significant pi-electron density. As expected, the incorporation of either 5-NapITP or 5-AnITP opposite templating DNA is reduced and presumably reflects steric constraints imposed by the large size of the polycyclic aromatic moieties. Furthermore, 5-NapITP is a chain terminator of translesion DNA synthesis because the DNA polymerase is unable to extend beyond the incorporated non-natural nucleotide. In addition, idle turnover measurements confirm that 5-NapIMP is stably incorporated opposite damaged DNA, and this enhancement reflects the overall favorable incorporation kinetic parameters coupled with a reduction in excision by the exonuclease-proofreading activity of the enzyme. On the basis of these data, we provide a comprehensive assessment of the potential role of pi-electron surface area for nucleotide incorporation opposite templating and nontemplating DNA catalyzed by the bacteriophage T4 DNA polymerase.

Lon protease, also known as protease La, is one of the simplest ATP-dependent proteases that plays vital roles in maintaining cellular functions by selectively eliminating misfolded, damaged and certain short-lived regulatory proteins. Although Lon is a homo-oligomer, each subunit of Lon contains both an ATPase and a protease active site. This relatively simple architecture compared to other hetero-oligomeric ATP-dependent proteases such as the proteasome makes Lon a useful paradigm for studying the mechanism of ATP-dependent proteolysis. In this article, we survey some recent developments in the mechanistic characterization of Lon with an emphasis on the utilization of pre-steady-state enzyme kinetic techniques to determine the timing of the ATPase and peptidase activities of the enzyme.

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.

Human glutaredoxin (GRx), also known as thioltransferase, is a 12 kDa thiol-disulfide oxidoreductase that is highly selective for reduction of glutathione-containing mixed disulfides. The apparent pK(a) for the active site Cys22 residue is approximately 3.5. Previously we observed that the catalytic enhancement by glutaredoxin could be ascribed fully to the difference between the pK(a) of its Cys22 thiol moiety and the pK(a) of the product thiol, each acting as a leaving group in the enzymatic and nonenzymatic reactions, respectively [Srinivasan et al. (1997), Biochemistry 36, 3199-3206]. Continuum electrostatic calculations suggest that the low pK(a) of Cys22 results primarily from stabilization of the thiolate anion by a specific ion-pairing with the positively charged Lys19 residue, although hydrogen bonding interactions with Thr21 also appear to contribute. Variants of Lys19 were considered to further assess the predicted role of Lys19 on the pK(a) of Cys22. The variants K19Q and K19L were generated by molecular modeling, and the pK(a) value for Cys22 was calculated for each variant. For K19Q, the predicted Cys22 pK(a) is 7.3, while the predicted value is 8.3 for K19L. The effects of the mutations on the interaction energy between the adducted glutathionyl moiety and GRx were roughly estimated from the van der Waals and electrostatic energies between the glutathionyl moiety and proximal protein residues in a mixed disulfide adduct of GRx and glutathione, i.e., the GRx-SSG intermediate. The values for the K19 mutants differed by only a small amount compared to those for the wild type enzyme intermediate. Together, the computational analysis predicted that the mutant enzymes would have markedly reduced catalytic rates while retaining the glutathionyl specificity displayed by the wild type enzyme. Accordingly, we constructed and characterized the K19L and K19Q mutants of two forms of the GRx enzyme. Each of the mutants retained glutathionyl specificity as predicted and displayed diminution in activity, but the decreases in activity were not to the extent predicted by the theoretical calculations. Changes in the respective Cys22-thiol pK(a) values of the mutant enzymes, as shown by pH profiles for iodoacetamide inactivation of the respective enzymes, clearly revealed that the K19-C22 ion pair cannot fully account for the low pK(a) of the Cys22 thiol. Additional contributions to stabilization of the Cys22 thiolate are likely donated by Thr21 and the N-terminal partial positive charge of the neighboring alpha-helix.

Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template, represents a common route toward mutagenesis and possibly disease development. To further define the mechanism of this promutagenic process, we synthesized and tested the enzymatic incorporation of two isosteric 5-substituted indolyl-2'deoxyriboside triphosphates opposite an abasic site. The catalytic efficiency for the incorporation of the 5-cyclohexene-indole derivative opposite an abasic site is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects a substantial increase in the k(pol) value (compare 25 versus 0.5 s(-1), respectively) as opposed to an influence on ground-state binding of either non-natural nucleotide. The faster k(pol) value for the 5-cyclohexene-indole derivative indicates that pi-electron density enhances the rate of the enzymatic conformational change step required for insertion opposite the abasic site. However, the kinetic dissociation constants for the non-natural nucleotides are identical and indicate that pi-electron density does not directly influence ground-state binding opposite the DNA lesion. Surprisingly, each non-natural nucleotide can be incorporated opposite natural templating bases, albeit with a greatly reduced catalytic efficiency. In this instance, the lower catalytic efficiency is caused by a substantial decrease in the k(pol) value rather than perturbations in ground-state binding. Collectively, these data indicate that the rate of the conformational change during translesion DNA synthesis depends on pi-electron density, while the enhancement in ground-state binding appears related to the size and shape of the non-natural nucleotide.

It is widely accepted that the dynamic behavior of DNA polymerases during translesion DNA synthesis is dependent upon the nature of the DNA lesion and the incoming dNTP destined to be the complementary partner. We previously demonstrated that 5-nitro-1-indolyl-2'-deoxyribose-5'-triphosphate, a nonnatural nucleobase possessing enhanced base-stacking abilities, can be selectively incorporated opposite an abasic site (Reineks, E. Z., and Berdis, A. J. (2004) Biochemistry 43, 393-404.). While the enhancement in insertion presumably reflected the contributions of the pi-electrons present in the nitro group, other physical parameters such as solvation capabilities, dipole moment, surface area, and shape could also contribute. To evaluate these possibilities, a series of 5-substituted indole triphosphates were synthesized and tested for enzymatic incorporation into normal and damaged DNA by the bacteriophage T4 DNA polymerase. The overall catalytic efficiency for the insertion of the 5-phenyl-indole derivative opposite an abasic site is several orders of magnitude greater than the insertion of either the 5-fluoro- or the 5-amino-indole derivative. The generated structure-activity relationship indicates that pi-electrons play the largest role in modulating the catalytic efficiency for insertion opposite this nontemplating DNA lesion. Despite the large size of 5-phenyl-indole, the catalytic efficiency for its insertion opposite natural nucleobases is equal to or greater than that of the 5-fluoro- or 5-amino-indole derivatives. The higher catalytic efficiency reflects a higher binding affinity of 5-phenyl-1-indolyl-2'-deoxyribose-5'-triphosphate and suggests that the polymerase relies on pi-electron surface area rather than shape complementarity as a driving force for polymerization efficiency.

This manuscript reports the development of nonnatural nucleotide analogues that are preferentially incorporated opposite an abasic site, a common form of DNA damage. Competition experiments confirm that all of the nonnatural nucleotides tested are poorly incorporated into unmodified DNA. However, two analogues that contain extensive pi-electron density (5-nitro-indolyl-2'deoxyriboside triphosphate (5-NITP) and 5-phenyl-indolyl-2'deoxyriboside triphosphate (5-PhITP)) are selectively inserted opposite an abasic site and can prevent the incorporation of natural dNTPs. We demonstrate that the DNA polymerase is unable to extend beyond the incorporated nonnatural nucleotide, a result that provides direct evidence for their unique chain termination capabilities. Furthermore, these nonnatural analogues are more slowly excised once inserted opposite the DNA lesion compared to natural dNTPs. The rate of excision becomes significantly faster when the nonnatural analogues are paired opposite natural templating positions, a result that provides additional evidence for their preferential insertion opposite the DNA lesion. Moreover, idle turnover measurements confirm that the bacteriophage T4 polymerase more stably incorporates 5-NIMP and 5-PhIMP opposite damaged DNA compared to natural dNTPs. The reduced idle turnover of these analogues reflects favorable insertion kinetics coupled with reduced exonuclease-proofreading capacity. Collectively, these data demonstrate the ability to selectively inhibit translesion DNA synthesis in vitro. A novel strategy is proposed to potentially use these nucleoside analogues to enhance the chemotherapeutic effects of DNA damaging agents as well as a possible chemopreventive strategy to inhibit promutagenic DNA replication.

Previous pre-steady-state kinetic studies of equine infectious anemia virus-1 (EIAV) reverse transcriptase (RT) showed two effects of DNA substrates containing the central termination sequence (CTS) on the polymerization reaction: reduction of burst amplitude in single nucleotide addition experiments and accumulation of termination products during processive DNA synthesis [Berdis, A. J., Stetor, S. R., Le Grice, S. F. J., and Barkley, M. D. (2001) Biochemistry 40, 12140-12149]. The present study of HIV RT uses pre-steady-state kinetic techniques to evaluate the molecular mechanisms of the lower burst amplitudes using both random sequence and CTS-containing DNA substrates. The effects of various factors, including primer/template length, binding orientation, and protein concentration, on the burst amplitude were determined using random sequence DNA substrates. The percent active RT increases with total RT concentration, indicating that reversible dissociation of RT dimer is responsible for substoichiometric burst amplitudes with normal substrates. This finding was confirmed by gel mobility shift assays. Like EIAV RT, HIV RT showed lower burst amplitudes on CTS-containing DNA substrates compared to random sequences. The dissociation kinetics of RT-DNA complexes were monitored by enzyme activity and fluorescence. Biphasic kinetics were observed for both random sequence and CTS-containing DNA complexes, revealing two forms of the RT-DNA complex. A mechanism is proposed to account for reduction in burst amplitude of CTS-containing DNA that is consistent with the results of both single nucleotide addition and dissociation experiments. The two forms of the RT-DNA complex may represent partitioning of primer/template between the P- and N-sites on RT for the nucleic acid substrate.

Escherichia coli Lon, also known as protease La, is an oligomeric ATP-dependent protease, which functions to degrade damaged and certain short-lived regulatory proteins in the cell. To investigate the kinetic mechanism of E. coli Lon protease, we performed the first pre-steady-state kinetic characterization of the ATPase and peptidase activities of this enzyme. Using rapid quench-flow and fluorescence stopped-flow spectroscopy techniques, we demonstrated that ATP hydrolysis occurs before peptide cleavage, with the former reaction displaying a burst and the latter displaying a lag in product production. The detection of burst kinetics in ATP hydrolysis is indicative of a step after nucleotide hydrolysis being rate-limiting in ATPase turnover. At saturating substrate concentrations, the lag rate constant for peptide cleavage is comparable to the kcat of ATPase, indicating that two hydrolytic processes are coordinated during the first enzyme turnover. The involvement of subunit interaction during enzyme catalysis was detected as positive cooperativity in the binding and hydrolysis of substrates, as well as apparent asymmetry in the ATPase activity in Lon. When our data are taken together, they are consistent with a reaction model in which ATP hydrolysis is used to generate an active enzyme form that hydrolyzes peptide.

DNA polymerases catalyze the insertion of a nucleoside triphosphate into the growing polymer chain using the template strand as a guide. Numerous factors such as hydrogen bonding interactions, base-stacking contributions, and desolvation play important roles in controlling the efficiency and fidelity of this process. We previously demonstrated that 5-nitro-indolyl-2'-deoxyriboside triphosphate, a non-natural nucleobase with enhanced base-stacking properties, was more efficiently inserted opposite a non-templating DNA lesion compared to natural templating nucleobases (E. Z. Reineks and A. J. Berdis, Biochemistry, 2004, 43, 393-404). The catalytic enhancement was proposed to reflect increased base-stacking interactions of the non-natural nucleobase with the polymerase and DNA. However, the effects of desolvation could not be unambiguously refuted. To further address the contributions of base stacking and desolvation during translesion DNA replication, we synthesized indolyl-2'-deoxyriboside triphosphate, a nucleobase devoid of nitro groups, and measured its efficiency of enzymatic insertion into modified and unmodified DNA. Removal of the nitro group reduces the catalytic efficiency for insertion opposite an abasic site by 3600-fold. This results from a large decrease in the rate of polymerization (similar 450-fold) coupled with a modest decrease in binding affinity (similar 8-fold). Since both non-natural nucleobases show the same degree of hydrophobicity, we attribute this reduction to the loss of base-stacking contributions rather than desolvation capabilities. Indolyl-2'-deoxyriboside triphosphate can also be inserted opposite natural nucleobases. Surprisingly, the catalytic efficiency for insertion is nearly identical to that measured for insertion opposite an abasic site. These data are discussed within the context of pi-electron interactions of the incoming nucleobase with the polymerase:DNA complex. Despite this lack of insertion selectivity, the polymerase is unable to extend beyond the non-natural nucleobase. This result indicates that indolyl-2'-deoxyriboside triphosphate acts as an indiscriminate chain terminator of DNA synthesis that may have unique therapeutic applications.

Despite the nontemplating nature of the abasic site, dAMP is often preferentially inserted opposite the lesion, a phenomenon commonly referred to as the "A-rule". We have evaluated the molecular mechanism accounting for this unique behavior using a thorough kinetic approach to evaluate polymerization efficiency during translesion DNA replication. Using the bacteriophage T4 DNA polymerase, we have measured the insertion of a series of modified nucleotides and have demonstrated that increasing the size of the nucleobase does not correlate with increased insertion efficiency opposite an abasic site. One analogue, 5-nitroindolyl-2'-deoxyriboside triphosphate, was unique as it was inserted opposite the lesion with approximately 1000-fold greater efficiency compared to that for dAMP insertion. Pre-steady-state kinetic measurements yield a kpol value of 126 s(-1) and a Kd value of 18 microM for the insertion of 5-nitroindolyl-2'-deoxyriboside triphosphate opposite the abasic site. These values rival those associated with the enzymatic formation of a natural Watson-Crick base pair. These results not only reiterate that hydrogen bonding is not necessary for nucleotide insertion but also indicate that the base-stacking and/or desolvation capabilities of the incoming nucleobase may indeed play the predominant role in generating efficient DNA polymerization. A model accounting for the increase in catalytic efficiency of this unique nucleobase is provided and invokes pi-pi stacking interactions of the aromatic moiety of the incoming nucleobase with aromatic amino acids present in the polymerase's active site. Finally, differences in the rate of 5-nitroindolyl-2'-deoxyriboside triphosphate insertion opposite an abasic site are measured between the bacteriophage T4 DNA polymerase and the Klenow fragment. These kinetic differences are interpreted with regard to the differences in various structural components between the two enzymes and are consistent with the proposed model for DNA polymerization.

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, k(off), of 1.1s(-1) while the wild-type replicase remains more stably associated at the site of DNA damage by virtue of a slower measured rate constant (k(off) 0.009s(-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 Mn(2+) for Mg(2+) 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.