Incorporation Of Nucleoside Analogs Research Articles

Non-canonical nucleosides: Biomimetic triphosphorylation, incorporation into mRNA and effects on translation and structure.

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non-canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS-CoV-2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7-deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency.

Transcription shapes DNA replication initiation to preserve genome integrity

BackgroundEarly DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.ResultsWe develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.ConclusionRNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular phosphorylation site.

Nucleoside analogs used in cancer chemotherapy and in treatment of virus infections are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active form. The phosphorylated nucleoside analogs are incorporated into DNA and cause cell death or inhibit viral replication. Cellular DNA is replicated both in the nucleus and in the mitochondria, and nucleoside analogs may interfere with DNA replication in both these subcellular locations. In the present study we created a cell model system where nucleoside analogs were phosphorylated, and thereby pharmacologically activated, in either the nucleus, cytosol, or mitochondria of cancer cells. The system was based on the reconstitution of deoxycytidine kinase (dCK)-deficient Chinese hamster ovary cells with genetically engineered dCK targeted to the different subcellular compartments. The nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, whereas the nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We further show that the nucleoside analogs phosphorylated in the mitochondria induced cell death by an apoptotic program. These data showed that the subcellular site of nucleoside analog phosphorylation is an important determinant for incorporation of nucleoside analogs into nuclear or mitochondrial DNA.

Visualization of Mitochondrial RNA Granules in Cultured Cells Using 5-Bromouridine Labeling.

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.

Asymmetric histone inheritance via strand-specific incorporation and biased replication fork movement.

SUMMARYMany stem cells undergo asymmetric division to produce a self-renewing stem cell and a differentiating daughter cell. Here we show that, similarly to H3, histone H4 is inherited asymmetrically in Drosophila melanogaster male germline stem cells undergoing asymmetric division. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy and chromatin fiber analyses with proximity ligation assays on intact nuclei, we find that old H3 is preferentially incorporated by the leading strand whereas newly synthesized H3 is enriched on the lagging strand. Using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional replication fork movement in testes-derived chromatin and DNA fibers. Biased fork movement coupled with a strand preference in histone incorporation would explain how asymmetric old and new H3 and H4 are established during replication. These results suggest a role for DNA replication in patterning epigenetic information in asymmetrically dividing cells in multicellular organisms.

Nucleoside analogs in the study of the epitranscriptome.

Over 150 unique RNA modifications are now known including several nonstandard nucleotides present in the body of messenger RNAs. These modifications can alter a transcript's function and are collectively referred to as the epitrancriptome. Chemically modified nucleoside analogs are poised to play an important role in the study of these epitranscriptomic marks. Introduced chemical features on nucleic acid strands provide unique structures or reactivity that can be used for downstream detection or quantification. Three methods are used in the field to synthesize RNA containing chemically modified nucleoside analogs. Nucleoside analogs can be introduced by metabolic labeling, via polymerases with modified nucleotide triphosphates or via phosphoramidite-based chemical synthesis. In this review, these methods for incorporation of nucleoside analogs will be discussed with specific recently published examples pertaining to the study of the epitranscriptome.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU.

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Asymmetric Histone Incorporation During DNA Replication in <i>Drosophila </i>Male Germline Stem Cells

Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. We previously reported that in male Drosophila germline stem cells (GSCs), preexisting (old) histone H3 is segregated to the self-renewed stem cell, whereas the daughter incorporates newly synthesized (new) H3. Here, we show that histone H4 is likewise segregated asymmetrically, while histones H2A and H2B are segregated symmetrically. Using superresolution imaging, we visualize spatially separable old and new H3 distributions in interphase GSC nuclei and on isolated replicating chromatin fibers. Furthermore, using both superresolved chromatin fibers and proximity ligation assay, we demonstrate that old H3 are preferentially retained on the leading strand while new H3 preferentially associate with the lagging strand. Finally, using a dual nucleoside analog incorporation assay, the temporal separation between leading strand and lagging strand synthesis is detectable. Together, these results demonstrate that the spatial and temporal asymmetries inherent to DNA replication may serve to bias histone incorporation, suggesting an unappreciated role for DNA replication in asymmetrically dividing cells.

Application of the microfluidic-assisted replication track analysis to measure DNA repair in human and mouse cells

Functional studies of the roles that DNA helicases play in human cells have benefited immensely from DNA fiber (or single molecule) technologies, which enable us to discern minute differences in behaviors of individual replication forks in genomic DNA in vivo. DNA fiber technologies are a group of methods that use different approaches to unravel and stretch genomic DNA to its contour length, and display it on a glass surface in order to immuno-stain nucleoside analog incorporation into DNA to reveal tracks (or tracts) of replication. We have previously adopted a microfluidic approach to DNA stretching and used it to analyze DNA replication. This method was introduced under the moniker maRTA or microfluidic-assisted Replication Track Analysis, and we have since used it to analyze roles of the RECQ helicases WRN and BLM, and other proteins in normal and perturbed replication. Here we describe a novel application of maRTA to detect and measure repair of DNA damage produced by three different agents relevant to etiology or therapy of cancer: methyl-methanesulfonate, UV irradiation, and mitomycin C. Moreover, we demonstrate the utility of this method by analyzing DNA repair in cells with reduced levels of WRN or of the base excision repair protein XRCC1.

Targeting the replisome with transduced monoclonal antibodies triggers lethal DNA replication stress in cancer cells

Although chemical inhibition of the DNA damage response (DDR) in cancer cells triggers cell death, it is not clear if the fork blockade achieved with inhibitors that neutralise proteins of the replisome is sufficient on its own to overcome the DDR. Monoclonal antibodies to PCNA, which block the DNA elongation process in vitro, have been developed. When these antibodies were transduced into cancer cells, they are able to inhibit the incorporation of nucleoside analogues. When co-delivered with anti-PCNA siRNA, the cells were flattened and the size of their nuclei increased by up to 3-fold, prior to cell death. Analysis of these nuclei by super-resolution microscopy revealed the presence of large numbers of phosphorylated histone H2AX foci. A senescence-like phenotype of the transduced cells was also observed upon delivery of the corresponding Fab molecules or following PCNA gene disruption or when the Fab fragment of an antibody that neutralises DNA polymerase alpha was used. Primary melanoma cells and leukaemia cells that are resistant to chemical inhibitors were similarly affected by these antibody treatments. These results demonstrate that transduced antibodies can trigger a lethal DNA replication stress, which kills cancer cells by abolishing the biological activity of several constituents of the replisome.

Research Techniques Made Simple: Techniques to Assess Cell Proliferation

Cell proliferation is commonly assayed in the laboratory for research purposes, but is increasingly used clinically to gauge tumor aggressiveness and potentially guide care. Therefore, both researchers and clinicians should have a basic understanding of techniques used to assess cell proliferation. Multiple cell proliferation assays exist, and the choice of method depends on the laboratory resources available, the types of cells/tissues to be studied, and the specific experimental goals. In this article, we identify four overarching categories of cell proliferation assays that signify various stages of the cell cycle: nucleoside-analog incorporation, cell cycle-associated protein detection, use of cytoplasmic proliferation dyes, and indirect measures of cell proliferation. Each method has strengths and limitations that should guide the dermatology investigator's choice of assay.

The sub-cellular localization of Sulfolobus DNA replication

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

Silencing of TEL/AML1 In Definitive Leukemic Cells Does Not Impair Cell Survival

AbstractAbstract 3229The most frequent structural chromosomal aberration in childhood acute lymphoblastic leukemia t(12;21) generates TEL/AML1 fusion gene. Resulting TEL/AML1 protein probably acts as an aberrant transcription factor that deregulates AML1-dependent transcription but its target genes and thus also the exact role in leukemic cells remain unknown. In vivo studies showed that TEL/AML1 itself is not sufficient to cause leukemia but may induce a preleukemic state characterized by the increased numbers of multipotent or B-cell progenitors with an incomplete block of differentiation. Despite its role for leukemia establishment the relevance of TEL/AML1 fusion gene for leukemia persistance has not been studied enough.To address this question and to explore the possibility of TEL/AML1-targeted therapy, we studied the effects of RNAi-mediated TEL/AML1 silencing on leukemic cells. As the only siRNA used for TEL/AML1 silencing published so far (Diakos et al, Blood, 2007) targets also the wild type AML1 (46% transcript reduction, our data), our first goal was to identify efficient and TEL/AML1-specific siRNA. We designed eleven different siRNAs spanning the fusion point of TEL/AML1 lacking the total sequence homology to wild type TEL and AML1 alleles to avoid their silencing. These 11 siRNAs were tested in HeLa cells transgenic for TEL/AML1-ires2-EGFP reporter. After lipofection into HeLa cells the efficiency of individual siRNAs was measured as a decrease of EGFP reporter fluorescence by flow cytometry. The best five siRNAs, that induced 50–58% silencing of the EGFP reporter, were tested at the mRNA level in TEL/AML-positive leukemic cell line. 24h after electroporation of siRNAs, when the silencing reached its maximum, two most efficient siRNAs induced 58% and 57% TEL/AML1 transcript reduction, respectively. We achieved 61% TEL/AML1 transcript reduction with the pool of both siRNAs while there was only slight reduction (14%) of wild type AML1 transcript. We used this efficient and specific siRNA pool to silence TEL/AML1 in REH and UOC-B6 TEL-AML1 positive cell lines and studied its effect on cell viability, proliferation and global gene expression. Applying two rounds of siRNA electroporation within 48 hours interval we achieved 74% and 86% TEL/AML1 protein knockdown in REH and UOC-B6 cells, respectively. We used trypan blue staining followed by optical microscopy to monitor cell viability and staining with annexin V and propidium idode to assess apoptosis rate by flow cytometry. Analysis of DNA content using staining with propidium iodide was performed to assess cell-cycle distribution. Incorporation of nucleoside analog was measured by flow cytometry to analyse de novo DNA synthesis as an indicator of proliferation rate. Despite the common expectation derived from studies on other fusion oncogenes (BCR/ABL, AML1/ETO, E2A/PBX), TEL/AML1 silencing neither decreased cell viability, nor induced apoptosis. On the contrary, TEL/AML1 depletion was accompanied by the slight but significant increase in the fraction of S-phase cells and corresponding rise in proliferation rate. Opposite effects on cell cycle distribution and proliferation were induced when we silenced wild type AML1. These findings support our hypothesis that TEL/AML1 may block previously established AML1 function in G1/S progression through the cell cycle. In line with the lack of effect on cell viability and discreet effect on cell-cycle distribution and proliferation we found no significant changes in global gene expression pattern upon TEL/AML1 depletion. Our data indicate, that TEL/AML1 is dispensable for the survival of definitive leukemic cells.This work was supported by grants MSM0021620813 and MZOFNM2005. Disclosures:No relevant conflicts of interest to declare.

Excision of nucleoside analogs in mitochondria by p53 protein

Nucleoside analogs, used against HIV, can be incorporated into a mitochondrial DNA by DNA polymerase gamma. Both the decrease in mitochondrial DNA and increased mutations of mitochondrial DNA may lead to mitochondrial diseases. The tumor suppressor protein p53 exhibits 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. In the present study, we investigated the ability of p53 to excise incorporated nucleoside analogs from DNA in mitochondria. The functional interaction of p53 and DNA polymerase gamma during the incorporation of nucleoside analog was examined in mitochondrial fractions of p53-null H1299 cells, as the source of DNA polymerase gamma. Primer extension reactions were carried out to elucidate the incorporation and removal of nucleoside analogs. The results demonstrate that the excision of incorporated nucleoside analogs in mitochondrial fractions of H1299 cells increased in the presence of purified recombinant p53, or cytoplasmic extracts of large cell carcinoma 2 cells expressing endogenous wild-type p53 (but not specifically predepleted extracts) or cytoplasmic extracts of H1299 cells overexpressing wild-type p53, but not exonuclease-deficient mutant p53-R175H. The amount of nucleoside analogs incorporated into the elongated DNA with mitochondrial fractions of human colon carcinoma 116 (HCT116)(p53+/+) cells was lower than that of HCT116(p53-/-) cells. Furthermore, mitochondrion-localized elevation of p53 in HCT116(p53+/+) cells, following the irradiation-stress stimuli, correlates with the reduction in incorporation of nucleoside analogs and wrong nucleotides. p53 in mitochondria may functionally interact with DNA polymerase gamma, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.

Characterization of cells expressing RNA polymerase II tagged with green fluorescent protein: Effect of ionizing irradiation on RNA synthesis

Purpose: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells.Materials and methods: We introduced an α-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with α-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1.Results: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional.Conclusions: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.

The incorporation of nucleoside analogs by human immunodeficiency virus type 1 reverse transcriptase decreases in the presence of polyamines

Nucleoside analogs (NAs) are an important class of anti-retroviral compounds used against human immunodeficiency virus (HIV). We have analyzed the potential effect of polyamines on the incorporation of NAs during DNA synthesis by HIV type-1 (HIV-1) reverse transcriptase (RT). The polyamines exert the ability to decrease the incorporation of various dideoxynucleoside triphosphates (ddATP, ddTTP or ddCTP) with both RNA/DNA and DNA/DNA substrates in the following order: spermine > spermidine > putrescine. The reduction is a sequence-independent effect, taking place at different sequence context. The results suggest that polyamines might affect the inhibition of reverse transcription by nucleoside analogs HIV-1 RT directed.

Excision of nucleoside analogs from DNA by p53 protein, a potential cellular mechanism of resistance to inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

We investigated the ability of p53 in cytoplasm to excise nucleoside analogs (NAs). A decrease in incorporation of NAs by human immunodeficiency virus type 1 reverse transcriptase and their excision from DNA by p53, provided by the cytoplasmic fraction of LCC2 cells, suggest that p53 in cytoplasm may act as an external proofreader for NA incorporation.

“Virostatics” as a Potential New Class of HIV Drugs

The combination of three or more antiretroviral drugs is referred to as highly active antiretroviral therapy (HAART) and constitutes the standard of care for HIV-1 patients in industrialized nations. Although HAART is usually effective in reducing viral load and re-constituting CD4 counts, latent virus reservoirs persist, and as many as 60 years therapy [1, 2] may be required to eradicate the virus. Meanwhile, patients are likely to experience drug related toxicity and may have to change therapy due to the emergence of drug resistant strains. For these reasons, the search for different therapeutic approaches continues. A new concept of antiviral/cytostatic ("virostatics") drugs has been proposed within the context of HAART to restrict virus target populations (CD4(+) T lymphocytes), target viral reservoirs, and possibly restore immune functions, by reducing excess immune activation, a fundamental component of HIV/AIDS pathogenesis. These virostatics include drugs such as hydroxyurea, mycophenolic acid, leflunomide and rapamycin, which are currently used for other therapeutic indications; and other experimental drugs, which are not for human use. They utilize multiple novel mechanisms of action to impede HIV by targeting host cellular proteins that are not susceptible to mutation. Therefore, their resistance profile appears to be quite favorable. Since many of these drugs act by inhibiting the synthesis of deoxynucleotides, essential for HIV reverse transcription, they favor the incorporation of nucleoside analogues into viral DNA, thus synergizing with the antiviral activity of currently used nucleoside reverse transcriptase inhibitors (NRTI). The rationale for the use of virostatics in HIV/AIDS, their mechanism of action, and ongoing preclinical and clinical research will be reviewed.

A Conformationally Constrained Nucleotide Analogue Controls the Folding Topology of a DNA G-Quadruplex

Guanine-rich DNA and RNA sequences can fold into unique structures known as G-quadruplexes. The structures of G-quadruplexes can be divided into several classes, depending on the parallel or antiparallel nature of the strands and the number of G-rich tracts present in an oligonucleotide. Oligonucleotides with single tracts of guanines form intermolecular parallel tetrameric G-quadruplexes. Oligonucleotides with two tracts of guanosines separated by two or more bases can form both intermolecular antiparallel fold-back dimeric and parallel tetrameric G-quadruplexes, and those with four tracts of guanosines can form both intramolecular parallel and antiparallel structures. Intramolecular G-qaudruplexes can fold into several folding topologies including antiparallel crossover basket, antiparallel chair, and parallel propeller. The ability to control the folding of G-quadruplexes would allow the physical, biochemical, and biological properties of these various folding topologies to be studied. Previously, the known methods to control the folding topology of G-quadruplexes included changing the buffer by varying the mono- and divalent cations that are present, and by changing the DNA sequence. Because the glycosidic bonds in the G-quartets of G-quadruplexes with parallel strands are in the anti conformation, we reasoned that incorporation of nucleoside analogues that prefer the anti conformation of the glycosidic bond into G-rich sequences would increase the preference for parallel G-quadruplex formation. As predicted, by positioning the conformationally constrained nucleotide analogue 2'-O-4'-C-methylene-linked ribonucleotide into specific positions of a DNA G-quadruplex we were able to shift the thermodynamically favored structure of a G-quadruplex from an antiparallel to a parallel structure.

Defining a Molecular Mechanism of Synergy between Nucleoside and Nonnucleoside AIDS Drugs

Combination therapies treating human immunodeficiency virus type 1 (HIV-1) infection delay the emergence of drug-resistant virus and exhibit synergistic inhibition. This synergy is observed within the two classes of inhibitors that target the essential viral reverse transcriptase (RT): the chain-terminating nucleoside analogs (NRTIs) and the allosteric nonnucleosides (NNRTIs) that bind in a pocket distinct from the active site. A general mechanism to define the molecular basis for synergy between these two classes remains to be elucidated. Previous mechanistic studies from our laboratory (Spence, R. A., Kati, W. M., Anderson, K. S., and Johnson, K. A. (1995) Science 267, 988-993) have shown that the natural deoxynucleoside triphosphate and the NNRTI can simultaneously bind to their respective sites. This work also suggests communication between the two sites, since the inhibition of RT by NNRTIs is manifested through a remote effect on the chemical step. This interplay between the two sites offers a plausible hypothesis for understanding synergy in which binding of NNRTIs modulates the chain termination by NRTIs. The present study supports this hypothesis by illustrating that the clinically approved NNRTIs, nevirapine and efavirenz, inhibit the ATP-mediated removal of AZTMP, d4TMP, ddCMP, (-)3TCMP, (-)FTCMP, and (+)3TCMP, thereby prolonging the effectiveness of chain termination. This inhibition is mediated through an effect on both the rate of the chemical step and binding of ATP, resulting in an overall decrease in efficiency of removal. This work substantiates communication between the two binding pockets, the sustained use of combination therapy to treat HIV infection, and a molecular basis for understanding synergy.