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As an important precursor for DNA synthesis, the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are necessary raw materials for DNA replication, recombination, and repair in cells. The correct synthesis and integrity of DNA are important manifestations of the genome stability, so the stability of the dNTP library state is essential to maintain the stability of the genome and the cell. In terms of the quality of the dNTP library, the incorporation of some heterogeneous dNTPs, such as oxidized dNTPs, into DNA can easily cause base substitutions and even DNA breaks and rearrangements, which will greatly damage the stability of the genome. At the same time, the cell has also evolved the corresponding NTP pyrophosphatase to remove it, and to correct the damaged DNA and repair the DNA gap by forming a DNA damage repair network. In terms of the number of dNTP libraries, the imbalance of the dNTP concentration and ratio will also cause base and frameshift mutations, which will also cause genome instability. As a result, cells have evolved a huge enzyme-controlled network to carry them out under precise control. This article mainly reviews the potential harm of damage to dNTP library components in cells, the clearance of damaged dNTPs, the regulation on the balance between dNTP library components, and finally discusses clinical diseases related to dNTP library homeostasis. It provides insights on the research of the correlation between the stability of the cellular dNTP library and the genome, and finally provides some theoretical basis for the treatment of related diseases.

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1 , which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions., (© 2022 Batté et al.)

Stimulated by the growing interest in the role of dNTP pools in physiological and malignant processes, we established dNTPpoolDB, the database that offers access to quantitative data on dNTP pools from a wide range of species, experimental and developmental conditions (https://dntppool.org/). The database includes measured absolute or relative cellular levels of the four canonical building blocks of DNA and of exotic dNTPs, as well. In addition to the measured quantity, dNTPpoolDB contains ample information on sample source, dNTP quantitation methods and experimental conditions including any treatments and genetic manipulations. Functions such as the advanced search offering multiple choices from custom-built controlled vocabularies in 15 categories in parallel, the pairwise comparison of any chosen pools, and control-treatment correlations provide users with the possibility to quickly recognize and graphically analyse changes in the dNTP pools in function of a chosen parameter. Unbalanced dNTP pools, as well as the balanced accumulation or depletion of all four dNTPs result in genomic instability. Accordingly, key roles of dNTP pool homeostasis have been demonstrated in cancer progression, development, ageing and viral infections among others. dNTPpoolDB is designated to promote research in these fields and fills a longstanding gap in genome metabolism research., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sterile alpha motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1), a dNTP triphosphohydrolase, regulates the levels of cellular dNTPs through their hydrolysis. SAMHD1 protects cells from invading viruses that depend on dNTPs to replicate and is frequently mutated in cancers and Aicardi-Goutières syndrome, a hereditary autoimmune encephalopathy. We discovered that SAMHD1 localizes at the immunoglobulin (Ig) switch region, and serves as a novel DNA repair regulator of Ig class switch recombination (CSR). Depletion of SAMHD1 impaired not only CSR but also IgH/c-Myc translocation. Consistently, we could inhibit these two processes by elevating the cellular nucleotide pool. A high frequency of nucleotide insertion at the break-point junctions is a notable feature in SAMHD1 deficiency during activation-induced cytidine deaminase-mediated genomic instability. Interestingly, CSR induced by staggered but not blunt, double-stranded DNA breaks was impaired by SAMHD1 depletion, which was accompanied by enhanced nucleotide insertions at recombination junctions. We propose that SAMHD1-mediated dNTP balance regulates dNTP-sensitive DNA end-processing enzyme and promotes CSR and aberrant genomic rearrangements by suppressing the insertional DNA repair pathway., (© 2020 The Authors.)

In this method, E. coli DNA Pol I binds to a nick or short gap in duplex DNA. The 5' → 3' exonuclease activity of Pol I then removes nucleotides from one strand of the DNA, creating a template for the synthesis of DNA by the 5' → 3' polymerase activity of Pol I. The simultaneous elimination of nucleotides from the 5' side and the addition of nucleotides to the 3' side result in movement of the nick (nick translation) along the DNA, which becomes labeled to high specific activity., (© 2020 Cold Spring Harbor Laboratory Press.)

Due to the extreme infectivity of Yersinia pestis it poses a serious threat as a potential biowarfare agent, which can be rapidly and facilely disseminated. A cost-effective and specific method for its rapid detection at extremely low levels is required, in order to facilitate a timely intervention for containment. Here, we report an ultrasensitive method exploiting a combination of isothermal nucleic acid amplification with a tailed forward primer and biotinylated dNTPs, which is performed in less than 30 min. The polymerase chain reaction (PCR) and enzyme linked oligonucleotide assay (ELONA) were used to optimise assay parameters for implementation on the LFA, and achieved detection limits of 45 pM and 940 fM using SA-HRP and SA-polyHRP, respectively. Replacing PCR with isothermal amplification, namely recombinase polymerase amplification, similar signals were obtained (314 fM), with just 15 min of amplification. The lateral flow detection of the isothermally amplified and labelled amplicon was then explored and detection limits of 7 fM and 0.63 fg achieved for synthetic and genomic DNA, respectively. The incorporation of biotinylated dNTPs and their exploitation for the ultrasensitive molecular detection of a nucleic acid target has been demonstrated and this generic platform can be exploited for a multitude of diverse real life applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G 1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G 1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Unlike activated CD4+ T cells, nondividing macrophages have an extremely small dNTP pool, which restricts HIV-1 reverse transcription. However, rNTPs are equally abundant in both of these cell types and reach much higher concentrations than dNTPs. The greater difference in concentration between dNTPs and rNTPs in macrophages results in frequent misincorporation of noncanonical rNTPs during HIV-1 reverse transcription. Here, we tested whether the highly abundant SAM domain- and HD domain-containing protein 1 (SAMHD1) deoxynucleoside triphosphorylase in macrophages is responsible for frequent rNTP incorporation during HIV-1 reverse transcription. We also assessed whether Vpx (viral protein X), an accessory protein of HIV-2 and some simian immunodeficiency virus strains that targets SAMHD1 for proteolytic degradation, can counteract the rNTP incorporation. Results from biochemical simulation of HIV-1 reverse transcriptase-mediated DNA synthesis confirmed that rNTP incorporation is reduced under Vpx-mediated dNTP elevation. Using HIV-1 vector, we further demonstrated that dNTP pool elevation by Vpx or deoxynucleosides in human primary monocyte-derived macrophages reduces noncanonical rNTP incorporation during HIV-1 reverse transcription, an outcome similarly observed with the infectious HIV-1 89.6 strain. Furthermore, the simian immunodeficiency virus mac239 strain, encoding Vpx, displayed a much lower level of rNTP incorporation than its ΔVpx mutant in macrophages. Finally, the amount of rNMPs incorporated in HIV-1 proviral DNAs remained unchanged for ∼2 weeks in macrophages. These findings suggest that noncanonical rNTP incorporation is regulated by SAMHD1 in macrophages, whereas rNMPs incorporated in HIV-1 proviral DNA remain unrepaired. This suggests a potential long-term DNA damage impact of SAMHD1-mediated rNTP incorporation in macrophages., (© 2020 Oo et al.)

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate β subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a Mn II 2 cluster by free superoxide to yield a metal-based Mn III Mn IV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive β subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.

The balance and the overall concentration of intracellular deoxyribonucleoside triphosphates (dNTPs) are important determinants of faithful DNA replication. Despite the established fact that changes in dNTP pools negatively influence DNA replication fidelity, it is not clear why certain dNTP pool alterations are more mutagenic than others. As intracellular dNTP pools are mainly controlled by ribonucleotide reductase (RNR), and given the limited number of eukaryotic RNR mutations characterized so far, we screened for RNR1 mutations causing mutator phenotypes in Saccharomyces cerevisiae. We identified 24 rnr1 mutant alleles resulting in diverse mutator phenotypes linked in most cases to imbalanced dNTPs. Among the identified rnr1 alleles the strongest mutators presented a dNTP imbalance in which three out of the four dNTPs were elevated (dCTP, dTTP and dGTP), particularly if dGTP levels were highly increased. These rnr1 alleles caused growth defects/lethality in DNA replication fidelity-compromised backgrounds, and caused strong mutator phenotypes even in the presence of functional DNA polymerases and mismatch repair. In summary, this study pinpoints key residues that contribute to allosteric regulation of RNR's overall activity or substrate specificity. We propose a model that distinguishes between different dNTP pool alterations and provides a mechanistic explanation why certain dNTP imbalances are particularly detrimental.

Background: Williams-Beuren syndrome (WBS) is caused by a microdeletion of chromosome arm 7q11.23. A rapid and inexpensive genotyping method to detect microdeletion on 7q11.23 needs to be developed for the diagnosis of WBS. This study describes the development of a new type of molecular diagnosis method to detect microdeletion on 7q11.23 based upon high-resolution melting (HRM)., Methods: Four genes on 7q11.23 were selected as the target genes for the deletion genotyping. dNTP-limited duplex PCR was used to amplify the reference gene, CFTR, and one of the four genes respectively on 7q11.23. An HRM assay was performed on the PCR products, and the height ratio of the negative derivative peaks between the target gene and reference gene was employed to analyze the copy number variation of the target region., Results: A new genotyping method for detecting 7q11.23 deletion was developed based upon dNTP-limited PCR and HRM, which cost only 96 min. Samples from 15 WBS patients and 12 healthy individuals were genotyped by this method in a blinded fashion, and the sensitivity and specificity was 100% (95% CI, 0.80-1, and 95% CI, 0.75-1, respectively) which was proved by CytoScan HD array., Significance: The HRM assay we developed is an rapid, inexpensive, and highly accurate method for genotyping 7q11.23 deletion. It is potentially useful in the clinical diagnosis of WBS., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Retroviral reverse transcriptase catalyses the synthesis of an integration-competent dsDNA molecule, using as a substrate the viral RNA. Using optical tweezers, we follow the Murine Leukemia Virus reverse transcriptase as it performs strand-displacement polymerization on a template under mechanical force. Our results indicate that reverse transcriptase functions as a Brownian ratchet, with dNTP binding as the rectifying reaction of the ratchet. We also found that reverse transcriptase is a relatively passive enzyme, able to polymerize on structured templates by exploiting their thermal breathing. Finally, our results indicate that the enzyme enters the recently characterized backtracking state from the pre-translocation complex., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Even small variations in dNTP concentrations decrease DNA replication fidelity, and this observation prompted us to analyze genomic cancer data for mutations in enzymes involved in dNTP metabolism. We found that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), a deoxyribonucleoside triphosphate triphosphohydrolase that decreases dNTP pools, is frequently mutated in colon cancers, that these mutations negatively affect SAMHD1 activity, and that several SAMHD1 mutations are found in tumors with defective mismatch repair. We show that minor changes in dNTP pools in combination with inactivated mismatch repair dramatically increase mutation rates. Determination of dNTP pools in mouse embryos revealed that inactivation of one SAMHD1 allele is sufficient to elevate dNTP pools. These observations suggest that heterozygous cancer-associated SAMHD1 mutations increase mutation rates in cancer cells.

The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Malyshev et al. showed that the four-letter genetic code within a living organism could be expanded to include the unnatural DNA bases dNaM and d5SICS. However, verification and detection of these unnatural bases in DNA requires new sequencing techniques. Here we provide proof of concept detection of dNaM and d5SICS in DNA oligomers via nanopore sequencing using the nanopore MspA. We find that both phi29 DNA polymerase and Hel308 helicase are capable of controlling the motion of DNA containing dNaM and d5SICS through the pore and that single reads are sufficient to detect the presence and location of dNaM and d5SICS within single molecules.

Regulation of intracellular deoxynucleoside triphosphate (dNTP) pool is critical to genomic stability and cancer development. Imbalanced dNTP pools can lead to enhanced mutagenesis and cell proliferation resulting in cancer development. Therapeutic agents that target dNTP synthesis and metabolism are commonly used in treatment of several types of cancer. Despite several studies, the molecular mechanisms that regulate the intracellular dNTP levels and maintain their homeostasis are not completely understood. The discovery of SAMHD1 as the first mammalian dNTP triphosphohydrolase provided new insight into the mechanisms of dNTP regulation. SAMHD1 maintains the homeostatic dNTP levels that regulate DNA replication and damage repair. Recent progress indicates that gene mutations and epigenetic mechanisms lead to downregulation of SAMHD1 activity or expression in multiple cancers. Impaired SAMHD1 function can cause increased dNTP pool resulting in genomic instability and cell-cycle progression, thereby facilitating cancer cell proliferation. This review summarizes the latest advances in understanding the importance of dNTP metabolism in cancer development and the novel function of SAMHD1 in regulating this process.

Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼ 50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol η(core) (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP >> dCTP and dTTP. Pol η(core) showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol η(core) bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol η(core) relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5'-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.

Macrophages are a major target cell for HIV-1, and their infection contributes to HIV pathogenesis. We have previously shown that the cyclin-dependent kinase inhibitor p21 inhibits the replication of HIV-1 and other primate lentiviruses in human monocyte-derived macrophages by impairing reverse transcription of the viral genome. In the attempt to understand the p21-mediated restriction mechanisms, we found that p21 impairs HIV-1 and simian immunodeficiency virus (SIV)mac reverse transcription in macrophages by reducing the intracellular deoxyribonucleotide (dNTP) pool to levels below those required for viral cDNA synthesis by a SAM domain and HD domain-containing protein 1 (SAMHD1)-independent pathway. We found that p21 blocks dNTP biosynthesis by down-regulating the expression of the RNR2 subunit of ribonucleotide reductase, an enzyme essential for the reduction of ribonucleotides to dNTP. p21 inhibits RNR2 transcription by repressing E2F1 transcription factor, its transcriptional activator. Our findings unravel a cellular pathway that restricts HIV-1 and other primate lentiviruses by affecting dNTP synthesis, thereby pointing to new potential cellular targets for anti-HIV therapeutic strategies.

The concentration of ribonucleoside triphosphates (rNTPs) in cells is far greater than the concentration of deoxyribonucleoside triphosphates (dNTPs), and this pool imbalance presents a challenge for DNA polymerases (Pols) to select their proper substrate. This report examines the effect of nucleotide pool imbalance on the rate and fidelity of the Escherichia coli replisome. We find that rNTPs decrease replication fork rate by competing with dNTPs at the active site of the C-family Pol III replicase at a step that does not require correct base-pairing. The effect of rNTPs on Pol rate generalizes to B-family eukaryotic replicases, Pols δ and ε. Imbalance of the dNTP pool also slows the replisome and thus is not specific to rNTPs. We observe a measurable frequency of rNMP incorporation that predicts one rNTP incorporated every 2.3 kb during chromosome replication. Given the frequency of rNMP incorporation, the repair of rNMPs is likely rapid. RNase HII nicks DNA at single rNMP residues to initiate replacement with dNMP. Considering that rNMPs will mark the new strand, RNase HII may direct strand-specificity for mismatch repair (MMR). How the newly synthesized strand is recognized for MMR is uncertain in eukaryotes and most bacteria, which lack a methyl-directed nicking system. Here we demonstrate that Bacillus subtilis incorporates rNMPs in vivo, that RNase HII plays a role in their removal, and the RNase HII gene deletion enhances mutagenesis, suggesting a possible role of incorporated rNMPs in MMR.

HIV-1 reverse transcriptase (RT) frequently incorporates ribonucleoside triphosphates (rNTPs) during proviral DNA synthesis, particularly under the limited dNTP conditions found in macrophages. We investigated the mechanistic impacts of an rNMP embedded in DNA templates on HIV-1 RT-mediated DNA synthesis. We observed that the template-embedded rNMP induced pausing of RT and delayed DNA synthesis kinetics at low macrophage dNTP concentrations but not at high T cell dNTP concentrations. Although the binding affinity of RT to the rNMP-containing template-primer was not altered, the dNTP incorporation kinetics of RT were significantly reduced at one nucleotide upstream and downstream of the rNMP site, leading to pause sites. Finally, HIV-1 RT becomes more error-prone at rNMP sites with an elevated mismatch extension capability but not enhanced misinsertion capability. Together these data suggest that rNMPs embedded in DNA templates may influence reverse transcription kinetics and impact viral mutagenesis in macrophages.

In normal human cells, oncogene-induced senescence (OIS) depends on induction of DNA damage response. Oxidative stress and hyperreplication of genomic DNA have been proposed as major causes of DNA damage in OIS cells. Here, we report that down-regulation of deoxyribonucleoside pools is another endogenous source of DNA damage in normal human fibroblasts (NHFs) undergoing HRAS(G12V)-induced senescence. NHF-HRAS(G12V) cells underexpressed thymidylate synthase (TS) and ribonucleotide reductase (RR), two enzymes required for the entire de novo deoxyribonucleotide biosynthesis, and possessed low dNTP levels. Chromatin at the promoters of the genes encoding TS and RR was enriched with retinoblastoma tumor suppressor protein and histone H3 tri-methylated at lysine 9. Importantly, ectopic coexpression of TS and RR or addition of deoxyribonucleosides substantially suppressed DNA damage, senescence-associated phenotypes, and proliferation arrest in two types of NHF-expressing HRAS(G12V). Reciprocally, short hairpin RNA-mediated suppression of TS and RR caused DNA damage and senescence in NHFs, although less efficiently than HRAS(G12V). However, overexpression of TS and RR in quiescent NHFs did not overcome proliferation arrest, suggesting that unlike quiescence, OIS requires depletion of dNTP pools and activated DNA replication. Our data identify a previously unknown role of deoxyribonucleotides in regulation of OIS., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a "steric-gate" to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker, and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3'-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs.

We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol β mutants.

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

Coordinated replication of eukaryotic nuclear genomes is asymmetric, with copying of a leading strand template preceding discontinuous copying of the lagging strand template. Replication is catalyzed by DNA polymerases α, δ and ɛ, enzymes that are related yet differ in physical and biochemical properties, including fidelity. Recent studies suggest that Pol ɛ is normally the primary leading strand replicase, whereas most synthesis by Pol δ occurs during lagging strand replication. New studies show that replication asymmetry can generate strand-specific genome instability resulting from biased deoxynucleotide pools and unrepaired ribonucleotides incorporated into DNA during replication, and that the eukaryotic replication machinery has evolved to most efficiently correct those replication errors that are made at the highest rates., (Published by Elsevier Ltd.)

Earlier, we postulated that Gln91 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) stabilizes the side chain of Tyr183 via hydrogen bonding interaction between O(H) of Tyr183 and CO of Q91 [Harris, D., et al. (1998) Biochemistry 37, 9630-9640]. To test this hypothesis, we generated mutant derivatives of Gln91 and analyzed their biochemical properties. The efficiency of reverse transcription was severely impaired by nonconservative substitution of Gln with Ala, while conservative substitution of Gln with Asn resulted in an approximately 70% loss of activity, a value similar to that observed with the Y183F mutation. The loss of polymerase activity from both Q91A and Q91N was significantly improved by a Met to Val substitution at position 184. Curiously, the Q91N mutant exhibited stringency in discriminating between correct and incorrect nucleotides, suggesting its possible interaction with residues influencing the flexibility of the dNTP binding pocket. In contrast, both double mutants, Q91A/M184V and Q91N/M184V, are found to be as error prone as the wild-type enzyme. We propose a model that suggests that subtle structural changes in the region due to mutation at position 91 may influence the stability of the side chain of Tyr183 in the catalytic YMDD motif of the enzyme, thus altering the active site geometry that may interfere in substrate recognition., (© 2011 American Chemical Society)

Ribonucleotide reduction provides deoxynucleotides for nuclear and mitochondrial (mt) DNA replication and DNA repair. In cycling mammalian cells the reaction is catalyzed by two proteins, R1 and R2. A third protein, p53R2, with the same function as R2, occurs in minute amounts. In quiescent cells, p53R2 replaces the absent R2. In humans, genetic inactivation of p53R2 causes early death with mtDNA depletion, especially in muscle. We found that cycling fibroblasts from a patient with a lethal mutation in p53R2 contained a normal amount of mtDNA and showed normal growth, ribonucleotide reduction, and deoxynucleoside triphosphate (dNTP) pools. However, when made quiescent by prolonged serum starvation the mutant cells strongly down-regulated ribonucleotide reduction, decreased their dCTP and dGTP pools, and virtually abolished the catabolism of dCTP in substrate cycles. mtDNA was not affected. Also, nuclear DNA synthesis and the cell cycle-regulated enzymes R2 and thymidine kinase 1 decreased strongly, but the mutant cell populations retained unexpectedly larger amounts of the two enzymes than the controls. This difference was probably due to their slightly larger fraction of S phase cells and therefore not induced by the absence of p53R2 activity. We conclude that loss of p53R2 affects ribonucleotide reduction only in resting cells and leads to a decrease of dNTP catabolism by substrate cycles that counterweigh the loss of anabolic activity. We speculate that this compensatory mechanism suffices to maintain mtDNA in fibroblasts but not in muscle cells with a larger content of mtDNA necessary for their high energy requirements.

We simulated in our laboratory a prebiotic environment where dry and wet periods were cycled. Under anhydrous conditions, lipid molecules present in the medium could form fluid lamellar matrices and work as organizing agents for the condensation of nucleic acid monomers into polymers. We exposed a mixture of 2'-deoxyribonucleoside 5'-monophosphates and a ssDNA oligomer template to this dry environment at 90 °C under a continuous gentle stream of CO(2) and we followed it with rehydration periods. After five dry/wet cycles we were able to detect the presence of a product that was complementary to the template. The reaction had a 0.5% yield with respect to the template, as measured by staining with the Pico Green(®) fluorescent probe. Absent initial template, the product of the reaction remained below the detection limit. In order to characterize the fidelity of replication, the synthesized strand was ligated to adapters, amplified by PCR, and sequenced. The alignment of the sequenced DNA to the expected complementary sequence revealed that the misincorporation rate was 9.9%. We present these results as a proof of concept for the possibility of having non-enzymatic transfer of sequence information in a prebiotically plausible environment., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

The introduction of a broad medical practice PCR "real time" is just beginning and dentistry is no exception. Modern molecular genetic methods provide numerous opportunities for diagnosis, assessment and prediction in patients with inflammatory periodontal diseases. Early and accurate diagnosis can allow in the future reduce the incidence of periodontitis and the progression of its course.

Inherent problems exist with sequencing-by-synthesis (SBS) methods which use fluorescein-labeled nucleotide incorporation into a target template based on a polymerase chain reaction (PCR). These problems include lowering the cost of sequencing and the removal of fluorescence in DNA sequencing for further reading. How can these sequencing problems be resolved? We present a sequencing strategy which we call an extension-quenching-extension sequencing on a microarray based on a two-primer hyperbranched rolling circle amplification (HRCA). The microarray is a high throughput and low-cost tool. The template on a microarray for SBS was prepared by HRCA. The Cy 5-labeled deoxyribonucleoside triphosphate (dNTP) species were incorporated in the extension reactions. We discovered that copper (CuSO(4)) can quench the fluorescence in DNA sequencing because it exhibits an energy-transfer mechanism of quenching from the fluorescein to the bound Cu(2+) ion. The fluorescein label needs to be destroyed after the readout by CuSO(4) before further reading is possible. This paper describes the process used which discovered a successful combination of temperature, concentration, and duration of use for CuSO(4) which successfully quenched the fluorescence. In this experiment, we used a known sequence as a template in order to provide a strategy for sequencing., ((c) 2009 Elsevier B.V. All rights reserved.)

Elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (AGEs) of proteins, lipids, and DNA. The formation of DNA-AGEs assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. The principal DNA-AGE, N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG), is formed as a mixture of R and S isomers at both the polymer and monomer levels. In order to examine the miscoding potential of this adduct, oligonucleotides substituted with (R)- and (S)-CEdG and the corresponding triphosphates (R)- and (S)-CEdGTP were synthesized, and base-pairing preferences for each stereoisomer were examined using steady-state kinetic approaches. Purine dNTPs were preferentially incorporated opposite template CEdG when either the Klenow (Kf(-)) or Thermus aquaticus (Taq) polymerases were used. The Kf(-) polymerase preferentially incorporated dGTP, whereas Taq demonstrated a bias for dATP. Kf(-) incorporated purines opposite the R isomer with greater efficiency, but Taq favored the S isomer. Incorporation of (R)- and (S)-CEdGTP only occurred opposite dC and was catalyzed by Kf(-) with equal efficiencies. Primer extension from a 3'-terminal CEdG was observed only for the R isomer. These data suggest CEdG is the likely adduct responsible for the observed pattern of G transversions induced by exposure to elevated glucose or its alpha-oxoaldehyde decomposition product methylglyoxal. The results imply that CEdG within template DNA and the corresponding triphosphate possess different syn/anti conformations during replication which influence base-pairing preferences. The implications for CEdG-induced mutagenesis in vivo are discussed.

DNA-protein cross-links (DPCs) are unique among DNA lesions in their unusually bulky nature. The steric hindrance imposed by cross-linked proteins (CLPs) will hamper DNA transactions, such as replication and transcription, posing an enormous threat to cells. In bacteria, DPCs with small CLPs are eliminated by nucleotide excision repair (NER), whereas oversized DPCs are processed exclusively by RecBCD-dependent homologous recombination (HR). Here we have assessed the roles of NER and HR for DPCs in mammalian cells. We show that the upper size limit of CLPs amenable to mammalian NER is relatively small (8-10 kDa) so that NER cannot participate in the repair of chromosomal DPCs in mammalian cells. Moreover, CLPs are not polyubiquitinated and hence are not subjected to proteasomal degradation prior to NER. In contrast, HR constitutes the major pathway in tolerance of DPCs as judged from cell survival and RAD51 and gamma-H2AX nuclear foci formation. Induction of DPCs results in the accumulation of DNA double strand breaks in HR-deficient but not HR-proficient cells, suggesting that fork breakage at the DPC site initiates HR and reactivates the stalled fork. DPCs activate both ATR and ATM damage response pathways, but there is a time lag between two responses. These results highlight the differential involvement of NER in the repair of DPCs in bacterial and mammalian cells and demonstrate the versatile and conserved role of HR in tolerance of DPCs among species.

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)(2) is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5'-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3' (FL-AT-1) and 5'-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3' (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to approximately 200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.

Photoactivated DNA analogs of nucleotide excision repair (NER) substrates have been created that are 48-mer duplexes containing in internal positions pyrimidine nucleotides with bulky substituents imitating lesions. Fluorochloroazidopyridyl, anthracenyl, and pyrenyl groups introduced using spacer fragments at 4N and 5C positions of dCMP and dUMP were used as model damages. The gel retardation and photo-induced affinity modification techniques were used to study the interaction of modified DNA duplexes with proteins in HeLa cell extracts containing the main components of NER protein complexes. It is shown that the extract proteins selectively bind and form covalent adducts with the model DNA. The efficiency and selectivity of protein modification depend on the structure of used DNA duplex. Apparent molecular masses of extract proteins, undergoing modification, were estimated. Mutual influence of simultaneous presence of extract proteins and recombinant NER protein factors XPC-HR23B, XPA, and RPA on interaction with the model DNA was analyzed. The extract proteins and RPA competed for interaction with photoactive DNA, mutually decreasing the yield of modification products. In this case the presence of extract proteins at particular concentrations tripled the increase in yield of covalent adducts formed by XPC. It is supposed that the XPC subunit interaction with DNA is stimulated by endogenous HR23B present in the extract. Most likely, the mutual effect of XPA and extract proteins stimulating formation of covalent adducts with model DNA is due to the interaction of XPA with endogenous RPA of the extract. A technique based on the use of specific antibodies revealed that RPA present in the extract is a modification target for photoactive DNA imitating NER substrates.

The ability to measure molar concentrations of deoxyribonucleoside 5'-triphosphates (dNTPs) within the mitochondrial matrix is important for several reasons. First, the spontaneous mutation rate for the mitochondrial genome is much higher than that for the nuclear genome, and dNTP concentrations are known determinants of DNA replication fidelity. Second, several human mitochondrial diseases involve perturbations of nucleotide metabolism, and dNTP pool analysis can help us to understand the consequences of these abnormalities. Third, it is important to understand how mtDNA is supplied with precursors in non-cycling cells, where the cytosolic machinery that supplies dNTPs for nuclear replication is downregulated. Fourth, the toxicity of several antiviral nucleoside analogs involves their metabolic activation within mitochondria, and dNTP pool analyses can help us to understand the processes leading to toxicity. Analyses of dNTP pools in whole-cell extracts from tissues or cultured cells are carried out either by HPLC or by an enzymatic method using DNA polymerase and defined templates. Because dNTP pools are much smaller in mitochondria than in whole cells, HPLC lacks the sensitivity needed for these measurements. The enzymatic method possesses sufficient sensitivity and is the method described in this chapter.

DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS), an autosomal recessive condition, is characterized by variable organ involvement with decreased mtDNA copy number and activities of respiratory chain enzymes in affected tissues. MtDNA depletion has been associated with mutations in nine autosomal genes, including thymidine kinase (TK2), which encodes a ubiquitous mitochondrial protein. To study the pathogenesis of TK2-deficiency, we generated mice harboring an H126N Tk2 mutation. Homozygous Tk2 mutant (Tk2(-/-)) mice developed rapidly progressive weakness after age 10 days and died between ages 2 and 3 weeks. Tk2(-/-) animals showed Tk2 deficiency, unbalanced dNTP pools, mtDNA depletion and defects of respiratory chain enzymes containing mtDNA-encoded subunits that were most prominent in the central nervous system. Histopathology revealed an encephalomyelopathy with prominent vacuolar changes in the anterior horn of the spinal cord. The H126N TK2 mouse is the first knock-in animal model of human MDS and demonstrates that the severity of TK2 deficiency in tissues may determine the organ-specific phenotype.

Our recent observation that hepatitis B virus (HBV) DNA polymerase (P) might initiate minus-strand DNA synthesis without primer [Kim et al., (2004) Virology 322, 22-30], raised a possibility that HBV P protein may have the potential to function as an RNA polymerase. Thus, we mutated Phe 436, a bulky amino acid with aromatic side chain, at the putative dNTP-binding cleft in reverse transcriptase (RT) domain of P protein to smaller amino acids (Gly or Val), and examined RNA polymerase activity. HBV core particles containing RT dNTP-binding cleft mutant P protein were able to incorporate (32)P-ribonucleotides, but not HBV core particles containing wild type (wt), priming-deficient mutant, or RT-deficient mutant P proteins. Since all the experiments were conducted with core particles isolated from transfected cells, our results indicate that the HBV RT mutant core particles containing RT dNTP-binding cleft mutant P protein could incorporate both deoxyribonucleotides and ribonucleotides in replicating systems.

The SWI/SNF chromatin remodeling complex plays a critical role in the coordination of gene expression with physiological stimuli. The synthetic enzymes ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase are coordinately regulated to ensure appropriate deoxyribonucleotide triphosphate levels. Particularly, these enzymes are actively repressed as cells exit the cell cycle through the action of E2F transcription factors and the retinoblastoma tumor suppressor/p107/p130 family of pocket proteins. This process is found to be highly dependent on SWI/SNF activity as cells deficient in BRG-1 and Brm subunits fail to repress these genes with activation of pocket proteins, and this deficit in repression can be complemented, via the ectopic expression of BRG-1. The failure to repress transcription does not involve a blockade in the association of E2F or pocket proteins p107 and p130 with promoter elements. Rather, the deficit in repression is due to a failure to mediate histone deacetylation of ribonucleotide reductase, dihydrofolate reductase, and thymidylate synthase promoters in the absence of SWI/SNF activity. The basis for this is found to be a failure to recruit mSin3B and histone deacetylase proteins to promoters. Thus, the coordinate repression of deoxyribonucleotide triphosphate metabolic enzymes is dependent on the action of SWI/SNF in facilitating the assembly of repressor complexes at the promoter.

A parallel 5'-d(TGGGGT)-3' quadruplex was formed in Na(+) solution and crystallized using lithium sulfate as the main precipitating agent. The X-ray structure was determined to 1.5 A resolution in space group P2(1) by molecular replacement. The asymmetric unit consists of a characteristic motif of two quadruplexes stacked at their 5' ends. All nucleotides are clearly defined in the density and could be positioned. A single bound Li(+) ion is observed at the surface of the column formed by the two joined molecules. Thus, this small alkali metal ion appears to be unsuitable as a replacement for the Na(+) ion in the central channel of G-quartets, unlike K(+) or Tl(+) ions. A well conserved constellation of water molecules is observed in the grooves of the dimeric structure.

In previous studies, it was reported that DNA fragments with the sequence d(gcGXYAgc) (where X = A or G and Y = A, T or G) form a stable base-intercalated duplex (Bi-duplex) in which the central X and Y residues are not involved in any base-pair interactions but are alternately stacked on each other between the two strands. To investigate the structural stability of the Bi-duplex, the crystal structure of d(gcGAACgc) with a point mutation at the sixth residue of the sequence, d(gcGAAAgc), has been determined. The two strands are associated in an antiparallel fashion to form two types of bulge-containing duplexes (Bc-duplexes), I and II, both of which are quite different from the Bi-duplex of the parent sequence. In both Bc-duplexes, three Watson-Crick G.C base pairs constitute the stem regions at the two ends. The A(4) residues are bulged in to form a pair with the corresponding A(4) residue of the opposite strand in either duplex. The A(4).A(4)* pair formation is correlated to the orientations of the adjacent A(5) residues. A remarkable difference between the two Bc-duplexes is seen at the A(5) residue. In Bc-duplex I, it is flipped out and comes back to interact with the G(3) residue. In Bc-duplex II, the A(5) residue extends outwards to interact with the G(7) residue of the neighbouring Bc-duplex I. These results indicate that trans sugar-edge/Hoogsteen (sheared-type) G(3).A(6)* base pairs are essential in the formation of a Bi-duplex of d(gcGXYAgc). On the other hand, the alternative conformations of the internal loops containing two consecutive bulged A residues suggest molecular switching.

We have previously reported the identification of a DNA repair system in Escherichia coli for the prevention of the stable incorporation of noncanonical purine dNTPs into DNA. We hypothesized that the RdgB protein is active on 2'-deoxy-N-6-hydroxylaminopurine triphosphate (dHAPTP) as well as deoxyinosine triphosphate. Here we show that RdgB protein and RdgB homologs from Saccharomyces cerevisiae, mouse, and human all possess deoxyribonucleoside triphosphate pyrophosphohydrolase activity and that all four RdgB homologs have high specificity for dHAPTP and deoxyinosine triphosphate compared with the four canonical dNTPs and several other noncanonical (d)NTPs. Kinetic analysis reveals that the major source of the substrate specificity lies in changes in K(m) for the various substrates. The expression of these enzymes in E. coli complements defects that are caused by the incorporation of HAP and an endogenous noncanonical purine into DNA. Our data support a preemptive role for the RdgB homologs in excluding endogenous and exogenous modified purine dNTPs from incorporation into DNA.