Recombination between gtfB and gtfC is required for survival of a dTDP-rhamnose synthesis-deficient mutant of Streptococcus mutans in the presence of sucrose.
(9/803)
The rml genes are involved in dTDP-rhamnose synthesis in Streptococcus mutans. A gene fusion between gtfB and gtfC, which both encode extracellular water-insoluble glucan-synthesizing enzymes, accompanied by inactivation of the rml genes was observed for cells grown in the presence of sucrose. The survival rates of rml mutants isolated in the absence of sucrose were drastically reduced in the presence of sucrose. The rates were consistent with the frequency of spontaneous gene fusions between gtfB and gtfC, suggesting that the spontaneous recombinant organisms were selected in the presence of sucrose. The rml mutants with a gtfB-gtfC fusion gene had markedly reduced water-insoluble glucan synthetic activity and lost the ability to colonize glass surfaces in the presence of sucrose. These results suggest that the rml mutants of S. mutans, which are defective in dTDP-rhamnose synthesis, can survive only in the absence of water-insoluble glucan synthesis. (+info)
Modulation of terminal deoxynucleotidyltransferase activity by the DNA-dependent protein kinase.
(10/803)
Rare Ig and TCR coding joints can be isolated from mice that have a targeted deletion in the gene encoding the 86-kDa subunit of the Ku heterodimer, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). However in the coding joints isolated from Ku86-/- animals, there is an extreme paucity of N regions (the random nucleotides added during V(D)J recombination by the enzyme TdT). This finding is consistent with a decreased frequency of coding joints containing N regions isolated from C.B-17 SCID mice that express a truncated form of the catalytic subunit of the DNA-PK (DNA-PKCS). This finding suggests an unexpected role for DNA-PK in addition of N nucleotides to coding ends during V(D)J recombination. In this report, we establish that TdT forms a stable complex with DNA-PK. Furthermore, we show that DNA-PK modulates TdT activity in vitro by limiting both the length and composition of nucleotide additions. (+info)
The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta.
(11/803)
A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site. (+info)
A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase.
(12/803)
Mutations in HIV-1 reverse transcriptase (RT) give rise to 3'-azido-3'-deoxythymidine (AZT) resistance by a mechanism that has not been previously reproduced in vitro. We show that mutant RT has increased ability to remove AZTMP from blocked primers through a nucleotide-dependent reaction, producing dinucleoside polyphosphate and extendible primer. In the presence of physiological concentrations of ATP, mutant RT extended 12% to 15% of primers past multiple AZTMP termination sites versus less than 0.5% for wild type. Although mutant RT also unblocked ddAMP-terminated primers more efficiently than wild-type RT, the removal of ddAMP was effectively inhibited by the next complementary dNTP (IC50 approximately equal to 12 microM). In contrast, the removal of AZTMP was not inhibited by dNTPs except at nonphysiological concentrations (IC50 > 200 microM). (+info)
Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation.
(13/803)
The Taq DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of Taq polymerase are deficient in two respects: (i) these enzymes incorporate each of the four dideoxynucleoside 5' triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (ii) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the Taq DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP. When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate. As a result, the G track generated during DNA sequencing by these Taq polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected. Another property of these Taq polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing. (+info)
Characterization of dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2.
(14/803)
The thymidine diphosphate-L-rhamnose biosynthesis pathway is required for assembly of surface glycoconjugates in a growing list of bacterial pathogens, making this pathway a potential therapeutic target. However, the terminal reactions have not been characterized. To complete assignment of the reactions, the four enzymes (RmlABCD) that constitute the pathway in Salmonella enterica serovar Typhimurium LT2 were overexpressed. The purified RmlC and D enzymes together catalyze the terminal two steps involving NAD(P)H-dependent formation of dTDP-L-rhamnose from dTDP-6-deoxy-D-xylo-4-hexulose. RmlC was assigned as the thymidine diphosphate-4-dehydrorhamnose 3,5-epimerase by showing its activity to be NAD(P)H-independent. Spectrofluorometric and radiolabeling experiments were used to demonstrate the ability of RmlC to catalyze the formation of dTDP-6-deoxy-L-lyxo-4-hexulose from dTDP-6-deoxy-D-xylo-4-hexulose. Under reaction conditions, RmlC converted approximately 3% of its substrate to product. RmlD was unequivocally identified as the thymidine diphosphate-4-dehydrorhamnose reductase. The reductase property of RmlD was shown by equilibrium analysis and its ability to enable efficient biosynthesis of dTDP-L-rhamnose, even in the presence of low amounts of dTDP-6-deoxy-L-lyxo-4-hexulose. Comparison of 23 known and predicted RmlD sequences identified several conserved amino acid residues, especially the serine-tyrosine-lysine catalytic triad, characteristic for members of the reductase/epimerase/dehydrogenase protein superfamily. In conclusion, RmlD is a novel member of this protein superfamily. (+info)
The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity.
(15/803)
Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in alpha-helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the two enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation. (+info)
Characterization of the activation pathway of phosphoramidate triester prodrugs of stavudine and zidovudine.
(16/803)
The phosphoramidate triester prodrugs of anti-human HIV 2', 3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2', 3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase. (+info)