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Christopher K. Mathews - One of the best experts on this subject based on the ideXlab platform.

  • Biochemistry and Biophysics presented on July 23, 1982. Title: DNA Precursor Compartmentation in Mammalian Cells: Metabolic and Antimetabolic Studies of Nuclear and Mitochondrial DNA Synthesis.
    2016
    Co-Authors: Redacted For Privacy, Christopher K. Mathews
    Abstract:

    This dissertation describes an investigation of DNA precursor supply for mitochondrial and nuclear DNA synthesis in HeLa cells and Mouse L-cells. HeLa cells were used for the quantitation of cellular and mitochondrial Deoxyribonucleoside Triphosphate (dNTP) and ribonucleoside Triphosphate (rNTP) pools and of changes in pools in response to treatment with the antimetabolites methotrexate (mtx) and 5-fluorodeoxyuridine (FUdR). Use of an enzymatic assay for dNTPs and of improved nucleotide extraction methods allowed quantitation of mitochondrial dNTP pools. All four mitochondrial dNTP pools expand following treatment with mtx or FUdR whereas cellular dTTP and dGTP pools are depleted. Mitochondrial rNTP pools were also found to expand in response to these antimetabolites. The cellular dGTP pool is severely depleted by mtx and FUdR ' which indicates dGTP levels might play an important role in mtx- and FUdR-induced cytotoxicity. Mouse L-cells were used to determine the relativ

  • nucleoside Triphosphate pool asymmetry in mammalian mitochondria
    Journal of Biological Chemistry, 2011
    Co-Authors: Linda J Wheeler, Christopher K. Mathews
    Abstract:

    Our laboratory has reported that Deoxyribonucleoside Triphosphate (dNTP) pools in rat tissue mitochondria are highly asymmetric, with dGTP predominating, and that the imbalance probably contributes toward the high spontaneous mutation rate of the mitochondrial genome. Ferraro et al. (Ferraro, P., Nicolosi, L., Bernardi, P., Reichard, P., and Bianchi, V. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 18586–18591) have challenged these findings, based upon their studies of mouse liver mitochondria. Moreover, they have identified a potential artifact in the DNA polymerase-based assay for dNTPs, based upon overestimation of dGTP when GTP levels in extracts are much higher than dGTP levels. We measured ribonucleoside Triphosphate (rNTP) pools in rat mitochondrial extracts and found that GTP pools exceed dGTP pools by 50-fold or less, not enough to interfere with the dGTP assay. Analysis of dNTP pools in state 3 mitochondria, after incubation with ADP and oxidizable substrates, gave similar results. We confirmed our earlier finding that rat mitochondrial dNTP pools are highly asymmetric. dNTP pools in cytosolic extracts are uniformly low, suggesting that the dNTP pool asymmetry arises within the mitochondrion. Moreover, we found rat tissue rNTP pools to be even more highly asymmetric, with ATP, for example, at least 2 orders of magnitude more abundant than CTP in liver extracts. This finding raises the possibility that transcription of the mitochondrial genome is more error-prone than transcription in the nucleus.

  • knockout of slc25a19 causes mitochondrial thiamine pyrophosphate depletion embryonic lethality cns malformations and anemia
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Marjorie J Lindhurst, Giuseppe Fiermonte, Shiwei Song, Eduard A Struys, Francesco De Leonardis, Pamela L Schwartzberg, Amy Chen, Alessandra Castegna, N M Verhoeven, Christopher K. Mathews
    Abstract:

    SLC25A19 mutations cause Amish lethal microcephaly (MCPHA), which markedly retards brain development and leads to α-ketoglutaric aciduria. Previous data suggested that SLC25A19, also called DNC, is a mitochondrial deoxyribonucleotide transporter. We generated a knockout mouse model of Slc25a19. These animals had 100% prenatal lethality by embryonic day 12. Affected embryos at embryonic day 10.5 have a neural-tube closure defect with ruffling of the neural fold ridges, a yolk sac erythropoietic failure, and elevated α-ketoglutarate in the amniotic fluid. We found that these animals have normal mitochondrial ribo- and Deoxyribonucleoside Triphosphate levels, suggesting that transport of these molecules is not the primary role of SLC25A19. We identified thiamine pyrophosphate (ThPP) transport as a candidate function of SLC25A19 through homology searching and confirmed it by using transport assays of the recombinant reconstituted protein. The mitochondria of Slc25a19−/− and MCPHA cells have undetectable and markedly reduced ThPP content, respectively. The reduction of ThPP levels causes dysfunction of the α-ketoglutarate dehydrogenase complex, which explains the high levels of this organic acid in MCPHA and suggests that mitochondrial ThPP transport is important for CNS development.

  • stimulation of mutagenesis by proportional Deoxyribonucleoside Triphosphate accumulation in escherichia coli
    DNA Repair, 2005
    Co-Authors: Linda J Wheeler, Indira Rajagopal, Christopher K. Mathews
    Abstract:

    Intracellular pool sizes of Deoxyribonucleoside Triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.

  • replication independent mcb gene induction and deoxyribonucleotide accumulation at g1 s in saccharomyces cerevisiae
    Journal of Biological Chemistry, 2003
    Co-Authors: Linda J Wheeler, Christopher K. Mathews, Gary F Merrill
    Abstract:

    In Saccharomyces cerevisiae, many genes encoding enzymes involved in deoxyribonucleotide synthesis are expressed preferentially near the G1/S boundary of the cell cycle. The relationship between the induction of deoxyribonucleotide-synthesizing genes, Deoxyribonucleoside Triphosphate levels, and replication initiation was investigated using factor-synchronized wild-type yeast or dbf4 yeast that are temperature-sensitive for replication initiation. Neither the timing nor extent of gene induction was inhibited when factor-arrested dbf4 cells were released into medium containing the ribonucleotide reductase inhibitor hydroxyurea, which blocks replication fork progression, or were released at 37 degrees C, which blocks replication origin firing. Thus, the induction of deoxyribonucleotide-synthesizing genes at G1/S was fully independent of DNA chain elongation or initiation. Deoxyribonucleoside Triphosphate levels increased severalfold at G1/S in wild-type cells and in dbf4 mutants incubated at the non-permissive temperature. Thus, Deoxyribonucleoside Triphosphate accumulation, like the induction of deoxyribonucleotide-synthesizing genes, was not dependent on replication initiation. Deoxyribonucleoside Triphosphate accumulation at G1/S was suppressed in cells lacking Swi6, a transcription factor required for normal cell cycle regulation of deoxyribonucleotide-synthesizing genes. The results suggest that cells use gene induction at G1/S as a mechanism to pre-emptively, rather than reflexively, increase the synthesis of DNA precursors to meet the demand of the replication forks for deoxyribonucleotides.

Michal Hocek - One of the best experts on this subject based on the ideXlab platform.

  • protected 2 Deoxyribonucleoside Triphosphate building blocks for the photocaging of epigenetic 5 hydroxymethyl cytosine in dna
    Organic and Biomolecular Chemistry, 2018
    Co-Authors: Soňa Bohacova, Michal Hocek, Zuzana Vanikova, Lenka Postova Slavětinska
    Abstract:

    2'-Deoxyribonucleoside Triphosphates (dNTPs) containing 5-(hydroxymethyl)cytosine (5hmC) protected with photocleavable groups (2-nitrobenzyl or 6-nitropiperonyl) were prepared and studied as substrates for the enzymatic synthesis of oligonucleotides and DNA containing a photocaged epigenetic 5hmC base. DNA probes containing photocaged or free 5hmC in the recognition sequence of restriction endonucleases were prepared and used for the study of the photorelease of caged DNA by UV or visible light at different wavelengths. The nitrobenzyl-protected dNTP was a slightly better substrate for DNA polymerases in primer extension or PCR, whereas the nitropiperonyl-protected nucleotide underwent slightly faster photorelease at 400 nm. However, both photocaged building blocks can be used in polymerase synthesis and the photorelease of 5hmC in DNA.

  • scope and limitations of the nicking enzyme amplification reaction for the synthesis of base modified oligonucleotides and primers for pcr
    Bioconjugate Chemistry, 2013
    Co-Authors: Petra Ménová, Veronika Raindlova, Michal Hocek
    Abstract:

    Enzymatic synthesis of short (10–22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified Deoxyribonucleoside Triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides.

  • Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR
    2013
    Co-Authors: Petra Ménová, Veronika Raindlová, Michal Hocek
    Abstract:

    Enzymatic synthesis of short (10–22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent­(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified Deoxyribonucleoside Triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides

Guohua Zhou - One of the best experts on this subject based on the ideXlab platform.

  • multiplex loop mediated isothermal amplification detection by sequence based barcodes coupled with nicking endonuclease mediated pyrosequencing
    Analytical Chemistry, 2012
    Co-Authors: Chao Liang, Yanan Chu, Sijia Cheng, Tomoharu Kajiyama, Hideki Kambara, Guohua Zhou
    Abstract:

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3′ end when the added Deoxyribonucleoside Triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogr...

  • Multiplex Loop-Mediated Isothermal Amplification Detection by Sequence-Based Barcodes Coupled with Nicking Endonuclease-Mediated Pyrosequencing
    2012
    Co-Authors: Chao Liang, Yanan Chu, Sijia Cheng, Tomoharu Kajiyama, Hideki Kambara, Guohua Zhou
    Abstract:

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3′ end when the added Deoxyribonucleoside Triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification

Beata G Vertessy - One of the best experts on this subject based on the ideXlab platform.

  • preventive dna repair by sanitizing the cellular deoxy nucleoside Triphosphate pool
    FEBS Journal, 2014
    Co-Authors: Gergely N Nagy, Ibolya Leveles, Beata G Vertessy
    Abstract:

    The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing Deoxyribonucleoside Triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-phosphorus atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

Chao Liang - One of the best experts on this subject based on the ideXlab platform.

  • multiplex loop mediated isothermal amplification detection by sequence based barcodes coupled with nicking endonuclease mediated pyrosequencing
    Analytical Chemistry, 2012
    Co-Authors: Chao Liang, Yanan Chu, Sijia Cheng, Tomoharu Kajiyama, Hideki Kambara, Guohua Zhou
    Abstract:

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3′ end when the added Deoxyribonucleoside Triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogr...

  • Multiplex Loop-Mediated Isothermal Amplification Detection by Sequence-Based Barcodes Coupled with Nicking Endonuclease-Mediated Pyrosequencing
    2012
    Co-Authors: Chao Liang, Yanan Chu, Sijia Cheng, Tomoharu Kajiyama, Hideki Kambara, Guohua Zhou
    Abstract:

    The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3′ end when the added Deoxyribonucleoside Triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification