Stringent steric exclusion mechanisms limit the misincorporation of ribonucleotides by high-fidelity DNA polymerases into genomic DNA. In contrast, low-fidelity Escherichia coli DNA polymerase V (pol V) has relatively poor sugar discrimination and frequently misincorporates ribonucleotides. Substitution of a steric gate tyrosine residue with alanine (umuC_Y11A) reduces sugar selectivity further and allows pol V to readily misincorporate ribonucleotides as easily as deoxynucleotides, whilst leaving its poor base-substitution fidelity essentially unchanged. However, the mutability of cells expressing the steric gate pol V mutant is very low due to efficient repair mechanisms that are triggered by the misincorporated rNMPs. Comparison of the mutation frequency between strains expressing wild-type and mutant pol V therefore allows us to identify pathways specifically directed at ribonucleotide excision repair (RER). We previously demonstrated that rNMPs incorporated by umuC_Y11A are efficiently removed from DNA in a repair pathway initiated by RNase HII. Using the same approach, we show here that mismatch repair and base excision repair play minimal back-up roles in RER in vivo. In contrast, in the absence of functional RNase HII, umuC_Y11A-dependent mutagenesis increases significantly in ΔuvrA, uvrB5 and ΔuvrC strains, suggesting that rNMPs misincorporated into DNA are actively repaired by nucleotide excision repair (NER) in vivo. Participation of NER in RER was confirmed by reconstituting ribonucleotide-dependent NER in vitro. We show that UvrABC nuclease-catalyzed incisions are readily made on DNA templates containing one, two, or five rNMPs and that the reactions are stimulated by the presence of mispaired bases. Similar to NER of DNA lesions, excision of rNMPs proceeds through dual incisions made at the 8th phosphodiester bond 5′ and 4th–5th phosphodiester bonds 3′ of the ribonucleotide. Ribonucleotides misinserted into DNA can therefore be added to the broad list of helix-distorting modifications that are substrates for NER.

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD′ 2 C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a trans activated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the ‘steric gate’ of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo . While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.

Escherichia coli pol V (UmuD′2C), the main translesion DNA polymerase, ensures continued nascent strand extension when the cellular replicase is blocked by unrepaired DNA lesions. Pol V is characterized by low sugar selectivity, which can be further reduced by a Y11A “steric-gate” substitution in UmuC that enables pol V to preferentially incorporate rNTPs over dNTPs in vitro. Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A in vitro, strains expressing umuC_Y11A exhibit low UV mutability and UV resistance. Here, we show that these phenotypes result from the concomitant dual actions of Ribonuclease HII (RNase HII) initiating removal of rNMPs from the nascent DNA strand and nucleotide excision repair (NER) removing UV lesions from the parental strand. In the absence of either repair pathway, UV resistance and mutagenesis conferred by umuC_Y11A is significantly enhanced, suggesting that the combined actions of RNase HII and NER lead to double-strand breaks that result in reduced cell viability. We present evidence that the Y11A-specific UV phenotype is tempered by pol IV in vivo. At physiological ratios of the two polymerases, pol IV inhibits pol V–catalyzed translesion synthesis (TLS) past UV lesions and significantly reduces the number of Y11A-incorporated rNTPs by limiting the length of the pol V–dependent TLS tract generated during lesion bypass in vitro. In a recA730 lexA(Def) ΔumuDC ΔdinB strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, umuC_Y11A-dependent spontaneous mutagenesis is only ∼7% of that observed with wild-type pol V, but increases to ∼39% of wild-type levels in an isogenic ΔrnhB strain and ∼72% of wild-type levels in a ΔrnhA ΔrnhB double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the E. coli genome, but at the cost of higher levels of cellular mutagenesis.

Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).In Escherichia coli two members of the Y family of polymerases are expressed as part of the inducible SOS response-DNA polymerase IV (Pol IV) and DNA Pol V. Both lack intrinsic proofreading activity and are considered low-fidelity DNA polymerases (14,32,33). Pol IV is encoded by the dinB gene (10,24,36). In normal cells, Pol IV is present at a relatively high level (ϳ250 molecules per cell) (13), compared to an estimated 30 molecules of the replicative Pol III (19). Upon SOS induction the Pol IV levels are increased by an additional 10-fold (13). The precise role of Pol IV under both normal and SOS-induced conditions is still under active investigation. In normal cells, the presence of Pol IV was shown to enhance the long-term survival and evolutionary fitness of E. coli (46). It has further been proposed that the major function of Pol IV is the restart of stalled replication forks (8). With regard to mutagenesis, a number of studies have indicated that Pol IV does not significantly affect the level of spontaneous mutations on the bacterial chromosome in growing cells (16,21,43), suggesting that it has limited access to the normal chromosomal growing point. On the other hand, Pol IV plays some role in mutagenesis on FЈ episomes (16) and contributes significantly to mutagenesis occurring in resting cells (adaptive mutagenesis) (6,20,35). Pol IV is able to carry out error-free or error-prone translesion synthesis, depending upon the nature of DNA damage and the sequence context (23), and has been shown to be involved in mutagenesis induced by 4-nitroquinoline-N-oxide, B[a]P-guanine adducts, and oxidative damage (9,13,17,23,30,38).DNA Pol V is a heterotrimeric complex (UmuDЈ 2 C) containing the umuC gene product, representing the catalytic subunit, and two copies of UmuDЈ, a RecA-produced proteolytic fragment of UmuD (28, 33). Pol V is required for most or all of SOS mutagenesis. Its intracellular concentration in normal cells is below the level of detection (Ͻ15 molecules per cell) (44). Upon full induction the umuDC operon produces approximately 2,400 molecules of UmuD and 200 molecules of UmuC. How many molecules of active Pol V are present under such conditions is an open question, as its components are tightly regulated both transcriptionally and posttranscriptionally (see reference 45 for a review). Pol V is proficient in bypassing UV-induced pyrimidine dimers and abasic sites in vitro (28,32,33), and this proficiency underlies its critical role in damage-induced mutagenesis in vivo.One interesting and experimentally informative aspect of the SOS response is the SOS mutator activity. This mutator activity is observed in strains that carry a constitutively activated RecA protein (e.g., RecA441 or RecA730) (34,40,41). Activati...

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The regulation of brain cytochrome P450 enzymes (CYPs) is different compared with respective hepatic enzymes. This may result from anatomical bases and physiological functions of the two organs. The brain is composed of a variety of functional structures built of different interconnected cell types endowed with specific receptors that receive various neuronal signals from other brain regions. Those signals activate transcription factors or alter functioning of enzyme proteins. Moreover, the blood-brain barrier (BBB) does not allow free penetration of all substances from the periphery into the brain. Differences in neurotransmitter signaling, availability to endogenous and exogenous active substances, and levels of transcription factors between neuronal and hepatic cells lead to differentiated expression and susceptibility to the regulation of CYP genes in the brain and liver. Herein, we briefly describe the CYP enzymes of CYP1-3 families, their distribution in the brain, and discuss brain-specific regulation of CYP genes. In parallel, a comparison to liver CYP regulation is presented. CYP enzymes play an essential role in maintaining the levels of bioactive molecules within normal ranges. These enzymes modulate the metabolism of endogenous neurochemicals, such as neurosteroids, dopamine, serotonin, melatonin, anandamide, and exogenous substances, including psychotropics, drugs of abuse, neurotoxins, and carcinogens. The role of these enzymes is not restricted to xenobiotic-induced neurotoxicity, but they are also involved in brain physiology. Therefore, it is crucial to recognize the function and regulation of CYP enzymes in the brain to build a foundation for future medicine and neuroprotection and for personalized treatment of brain diseases.