No abstract

No abstract

Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.The replication of both cellular and DNA virus genomes requires a balanced supply of deoxyribonucleoside triphosphates (dNTPs). In eukaryotic cells, conversion of ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates is catalyzed by ribonucleotide reductase (RNR), the rate-limiting enzyme in DNA precursor biosynthesis (56,60,61). Ribonucleotide reduction is the first of a series of metabolic reactions leading to DNA synthesis and as such is controlled at several levels. The same enzyme reduces all four ribonucleotides, and both substrate specificity and overall activity are tightly controlled by binding of NTP allosteric effectors. Substrate specificity is controlled by binding of ATP or dATP (CDP/UDP reduction), dTTP (GDP reduction), or dGTP (ADP reduction) to a specificity site in the R1 protein, while overall activity is controlled by binding ATP (activation) or dATP (inactivation) to an activity site (39). The activity of RNR is cell cycle regulated and is very low or not detectable in resting cells and maximal in S-phase cells (56,61). This is controlled both at the level of transcription and by regulation of protein stability (6,13,22,24).Three RNR classes have been characterized based on the mechanism for generation of the protein radical, metal cofactor requirement, and subunit composition (39). Human cells, like most eu...

Polymerase chain reaction (PCR) followed by reverse dot blot was used to detect Enterococcus faecalis leakage through mineral trioxide aggregate (MTA) apical obturations of pulpless teeth with open apices. Prepared root canals of 34 extracted teeth were given a standard apical foramen opening and received orthograde apical obturation with MTA; three groups had 1-, 2-, or 3-mm thickness. Sterilized specimens were inoculated with E. faecalis and incubated in sterile medium. DNA extracted from the specimens was amplified by polymerase chain reaction, which yielded a specific segment of E. faecalis 16S rDNA. On day 10 of incubation, no specimens were contaminated. On day 50, almost 17% of specimens were contaminated, with no statistically significant difference between groups (Chi-square = 0.48; df = 2; p = 0.787). Therefore, MTA provides an adequate seal even in cases of orthograde apical obturation of pulpless teeth with open apices.

No abstract