Intracellular organization of DNA precursor biosynthetic enzymes

Mathews, Christopher K.; Moen, Laura K.; Wang, Yeong; Sargent, R. Geoffrey

doi:10.1016/0968-0004(88)90182-x

Cited by 36 publications

(15 citation statements)

References 19 publications

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“…Enzymes involved in nucleotide metabolism are located mostly in a region where DNA synthesis occurs (34). Hence, the authentic E. coli enzyme may have specific structural features to facilitate proper subcellular localization.…”

Section: Table I Suppression Of E Coli Mutt Mutator Activity By Exprmentioning

confidence: 99%

Mouse MTH1 Protein with 8-Oxo-7,8-dihydro-2′-deoxyguanosine 5′-Triphosphatase Activity That Prevents Transversion Mutation

Kakuma

Nishida

Tsuzuki

et al. 1995

Journal of Biological Chemistry

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8-Oxo-7,8-dihydro-2-deoxyguanosine 5-triphosphate (8-oxo-dGTP) is formed in the nucleotide pool of a cell during normal cellular metabolism, and when it is incorporated into DNA causes mutation. Organisms possess 8-oxo-dGTPase, an enzyme that specifically degrades 8-oxo-dGTP to 8-oxo-dGMP. We isolated cDNA for mouse 8-oxo-dGTPase, using as a probe human MTH1 (Escherichia coli mutT homolog) cDNA. The nucleotide sequence of the cDNA revealed that the mouse MTH1 protein (molecular weight of 17,896) comprises 156 amino acid residues. When the cDNA for mouse 8-oxo-dGTPase was expressed in E. coli mutT ؊ mutant cells devoid of their own 8-oxo-dGTPase activity, an 18-kDa protein, which is cross-reactive with an anti-human MTH1 antibody, was formed. In such cells, the level of spontaneous mutation frequency that was elevated reverted to normal. High levels of 8-oxo-dGTPase activity were found in liver, thymus, and large intestine, whereas all other organs examined contained smaller amounts of the enzyme. In embryonic stem cells, an exceedingly high level of the enzyme was present.Oxygen radicals are generated during normal cellular metabolism, and the formation of such radicals is further enhanced by ionizing radiation and by various chemicals (1). Among many classes of DNA damage caused by oxygen radicals, an oxidized form of guanine base, 8-oxo-7,8-dihydroguanine (8-oxoguanine), 1 appears to play important roles in mutagenesis and in carcinogenesis (2-4). During DNA replication, 8-oxoguanine nucleotide can pair with cytosine and adenine nucleotides, with an almost equal efficiency, and transversion mutation ensues (5-7).Organisms are equipped with elaborate mechanisms to counteract such mutagenic effects of 8-oxoguanine. In Escherichia coli, two glycosylases encoded by the mutM and the mutY genes function to prevent mutation caused by 8-oxoguanine in DNA (8 -12). MutM protein removes 8-oxoguanine paired with cytosine and MutY protein removes adenine paired with 8-oxoguanine. Enzyme activities similar to those of MutM and MutY have been identified in mammalian cells (13)(14)(15)(16). A significant amount of 8-oxoguanine is formed in the chromosomal DNA of mammalian cells, and most damaged nucleotides are excised from the DNA and excreted into the urine (17). These enzymes may maintain spontaneous mutation frequency at certain low levels.Oxidation of guanine proceeds also in forms of free nucleotides, and an oxidized form of dGTP, 8-oxo-dGTP, is a potent mutagenic substrate for DNA synthesis (18). Organisms possess mechanisms for preventing mutation due to misincorporation of 8-oxo-dGTP. MutT protein of E. coli hydrolyzes 8-oxodGTP to the monophosphate, and lack of the mutT gene increases the occurrence of A:T to C:G transversion thousands of fold over the wild type level (18 -20). Human cells contain an enzyme similar to the MutT protein, and this enzyme specifically hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with a relatively low K m value, as compared with other deoxyribonucleoside triphosphates (21,22). Therefore, the hum...

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Section: Table I Suppression Of E Coli Mutt Mutator Activity By Exprmentioning

confidence: 99%

Mouse MTH1 Protein with 8-Oxo-7,8-dihydro-2′-deoxyguanosine 5′-Triphosphatase Activity That Prevents Transversion Mutation

Kakuma

Nishida

Tsuzuki

et al. 1995

Journal of Biological Chemistry

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“…Also, the dTYMK mRNA could be unstable in bacteria, or the human protein contains signals for its degradation or export out of E. coli. Furthermore, the human dTYMK protein may only be stable and active in supra-molecular complexes, such as the proposed "metabolon" multienzyme complex (22).…”

Section: Discussionmentioning

confidence: 99%

Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

Chaperon

2006

Appl Environ Microbiol

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This work reports the construction of Escherichia coli in-frame deletion strains of tmk, which encodes thymidylate kinase, Tmk. The tmk gene is located at the third position of a putative five-gene operon at 24.9 min on the E. coli chromosome, which comprises the genes pabC, yceG, tmk, holB, and ycfH. To avoid potential polar effects on downstream genes of the operon, as well as recombination with plasmid-encoded tmk, the tmk gene was replaced by the kanamycin resistance gene kka1, encoding amino glycoside 3-phosphotransferase kanamycin kinase. The kanamycin resistance gene is expressed under the control of the natural promoter(s) of the putative operon. The E. coli tmk gene is essential under any conditions tested. To show functional complementation in bacteria, the E. coli tmk gene was replaced by thymidylate kinases of bacteriophage T4 gp1, E. coli tmk, Saccharomyces cerevisiae cdc8, or the Homo sapiens homologue, dTYMK. Growth of these transgenic E. coli strains is completely dependent on thymidylate kinase activities of various origin expressed from plasmids. The substitution constructs show no polar effects on the downstream genes holB and ycfH with respect to cell viability. The presented transgenic bacteria could be of interest for testing of thymidylate kinase-specific phosphorylation of nucleoside analogues that are used in therapies against cancer and infectious diseases.

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“…This time is taken as a working value for the in vivo appearance of active ribonucleoside diphosphate reductase (13,45). This enzyme has been detected in extracts at about 5 min after infection (53), but its kinetics of formation is much different and much faster than the initial exponential rise in the rate of synthesis of the deoxyribonucleotides (13,45,51); the kinetics of the latter is presumed to be dependent on the rate of formation of the dNTP synthetase complex (11,34,41,52), which contains ribonucleotide reductase as a central component (the term dNTP synthetase complex is an amalgamation of earlier designations used in our publications and in those of C. K. Mathews and co-workers [1,11,34,45,51]). …”

Section: Discussionmentioning

confidence: 99%

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

Tseng

Hilfinger

et al. 1990

J Bacteriol

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We examined the expression of the bacteriophage T4 nrdA and nrdB genes, which encode the a2 and 02 subunits, respectively, of ribonucleoside diphosphate reductase, the first committed enzyme in the pathway of synthesis of the deoxyribonucleoside triphosphates. T4 nrdA, located 700 bp upstream from nrdB, has been shown previously to be transcribed by two major transcripts: a prereplicative, polycistronic message, Tu, originating at an immediate-early promoter, PEI that is 3.5 kb upstream from nrdA, and a postreplicative message commencing from a late promoter in its 5' flank. We have found a third promoter initiating a transcript at 159 nucleotides upstream from the reading frame of nrdB. PrdB functions only in the presence of the T4 motA gene product, which is required for middle (time) promoters, and therefore the onset of nrdB transcription is delayed more than 2 min after infection. Because of the distance of nrdA from PEI the inception of nrdA transcription (delayed early) coincides closely with that of nrdB. An apparent termination site, tA, occurs about 80 bp downstream from nrdA. Some of the polycistronic mRNA reading through the site after 5 min contributes to nrdB transcription. nrdA and nrdB genes in an uninfected host have been reported to be transcribed only coordinately. In contrast, T4 nrdA and nrdB are initially transcribed separately onto the PE and PnrdB transcripts, respectively, but at about 5 min after infection are transcribed both coordinately and on separate transcripts. Evidence is presented that Tu coordinately transcribes a deoxyribonucleotide operon in the order: frd, td, gene 'Y,' nrdA, nrdB. Since the 02 subunit is known to be formed after the a2 subunit, the expression of the nrdB gene determines the onset of deoxyribonucleoside triphosphate synthesis and thus of T4 DNA replication.

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Intracellular organization of DNA precursor biosynthetic enzymes

Cited by 36 publications

References 19 publications

Mouse MTH1 Protein with 8-Oxo-7,8-dihydro-2′-deoxyguanosine 5′-Triphosphatase Activity That Prevents Transversion Mutation

Mouse MTH1 Protein with 8-Oxo-7,8-dihydro-2′-deoxyguanosine 5′-Triphosphatase Activity That Prevents Transversion Mutation

Construction and Complementation of In-Frame Deletions of the Essential Escherichia coli Thymidylate Kinase Gene

Bacteriophage T4 nrdA and nrdB genes, encoding ribonucleotide reductase, are expressed both separately and coordinately: characterization of the nrdB promoter

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