Removal of Misincorporated Ribonucleotides from Prokaryotic Genomes: An Unexpected Role for Nucleotide Excision Repair

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 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts

The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts. June 4-7, 2019, Szczyrk, Polan

The Effect of Chronic Iloperidone Treatment on Cytochrome P450 Expression and Activity in the Rat Liver: Involvement of Neuroendocrine Mechanisms

In order to achieve a desired therapeutic effect in schizophrenia patients and to maintain their mental wellbeing, pharmacological therapy needs to be continued for a long time, usually from the onset of symptoms and for the rest of the patients’ lives. The aim of our present research is to find out the in vivo effect of chronic treatment with atypical neuroleptic iloperidone on the expression and activity of cytochrome P450 (CYP) in rat liver. Male Wistar rats received a once-daily intraperitoneal injection of iloperidone (1 mg/kg) for a period of two weeks. Twenty-four hours after the last dose, livers were excised to study cytochrome P450 expression (mRNA and protein) and activity, pituitaries were isolated to determine growth hormone-releasing hormone (GHRH), and blood was collected for measuring serum concentrations of hormones and interleukin. The results showed a broad spectrum of changes in the expression and activity of liver CYP enzymes, which are important for drug metabolism (CYP1A, CYP2B, CYP2C, and CYP3A) and xenobiotic toxicity (CYP2E1). Iloperidone decreased the expression and activity of CYP1A2, CP2B1/2, CYP2C11, and CYP3A1/2 enzymes but increased that of CYP2E1. The CYP2C6 enzyme remained unchanged. At the same time, the level of GHRH, GH, and corticosterone decreased while that of T3 increased, with no changes in IL-2 and IL-6. The presented results indicate neuroendocrine regulation of the investigated CYP enzymes during chronic iloperidone treatment and suggest a possibility of pharmacokinetic/metabolic interactions produced by the neuroleptic during prolonged combined treatment with drugs that are substrates of iloperidone-affected CYP enzymes

Role of DNA Polymerase IV in Escherichia coli SOS Mutator Activity

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)

Vitamin C in medicine and cosmetology

Ascorbic acid is one of the strongest antioxidants. It has a protective function against oxidative stress intracellularly. It fights free radicals and eliminates the effects of ultraviolet radiation. The human body is unable to synthesize vitamin C itself. Vitamin C meets supports the mechanism of removing toxins from the human body and has anticancer activity. Over the years, many studies have been carried out to confirm the beneficial effect of preventive and supportive action of cytostatics in the treatment of cancer. Clinical studies described in the study indicate that vitamin C used in combination with a cytostatic drug - gemcystabine, shows no side effects. Vitamin C applied topically on the skin shows a number of positive effects. Studies have shown that vitamin C accumulates in the deeper layers of the skin for about three days. Ascorbic acid has a protective effect against harmful ultraviolet radiation. Manufacturers of cosmetics often add ascorbic acid to cosmetics with sunscreens. In addition, vitamin C acts depigmenting. In order to increase the brightening effect, vitamin C is often combined with glycolic acid or retinol. Ascorbic acid is also used in anti-aging preparations due to the high ability of collagen synthesis in the skin. In topical preparations, ascorbic acid has hydrophilic properties - it is soluble in water and insoluble in fats. Hydrophilic ascorbic acid in synergy with lipophilic tocopherol reduces cell apoptosis and thymine dimer formation. Its stability improves the presence of stabilizing antioxidants, such as vitamin E and ferulic acid. For the preparation to retain its properties, it should be protected from light and oxygen. The development of biotechnology allows the closure of a micronised vitamin C in nanosomes and liposomes, which increases the ability to penetrate deep into the skin. A number of vitamin C derivatives such as ascorbyl tetrapalmitate and ascorbyl phosphate magnesium help achieve the desired results in topical application

Role of Escherichia coli DNA Polymerase IV in In Vivo Replication Fidelity

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

Mutator Phenotype Resulting from DNA Polymerase IV Overproduction in Escherichia coli: Preferential Mutagenesis on the Lagging Strand

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

Mechanisms Employed by <em>Escherichia coli</em> to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V

<div><p><em>Escherichia coli</em> 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 <em>in vitro.</em> Despite efficient error-prone translesion synthesis catalyzed by UmuC_Y11A <em>in vitro</em>, strains expressing <em>umuC</em>_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 <em>umuC</em>_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 <em>in vivo</em>. 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 <em>in vitro</em>. In a <em>recA730 lexA</em>(Def) Δ<em>umuDC</em> Δ<em>dinB</em> strain, plasmid-encoded wild-type pol V promotes high levels of spontaneous mutagenesis. However, <em>umuC</em>_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 Δ<em>rnhB</em> strain and ∼72% of wild-type levels in a Δ<em>rnhA</em> Δ<em>rnhB</em> double mutant. Our observations suggest that errant ribonucleotides incorporated by pol V can be tolerated in the <em>E. coli</em> genome, but at the cost of higher levels of cellular mutagenesis.</p> </div

<i>In vitro</i> translesion synthesis past a TT-CPD lesion catalyzed by mixtures of pol IV and pol V.

<p>Translesion DNA synthesis was performed using a circular DNA template with a running-start primer with its 3′ end located 5 bases before the 3′T of the CPD. Primer extension reactions catalyzed by pol IV (80 nM, lane 1 or 800 nM, lane 6), wild-type pol V (80 nM, lanes 2 and 7), pol V (UmuC_Y11A) (60 nM, lanes 4 and 9), or a combination of pol IV (80 nM, lane 3 and 5 or 800 nM, lane 8 and 10) with either wild-type (80 nM, lanes 3 and 8) or polV (UmuC_Y11A) (60 nM, lanes 5 and 10) were performed for 30 sec as described in the Methods section. Part of the template sequence and position of the gel wells and a CPD lesion are indicated to the right of the gel panel. As clearly observed, when present in a 10-fold excess (similar to SOS induced conditions), pol IV inhibits TLS catalyzed by pol V.</p

Model for the effect of RNase HII and NER proteins on UV sensitivity of strains proficient for ribonucleotide incorporation.

<p>Translesion replication catalyzed by Y11A mutant produces a TLS tract containing multiple ribonucleotides. NER excises UV-induced lesions and produces gaps on the template strand. RNase HII initiating removal of multiple rNMPs incorporated during TLS produces nicks on the daughter strand. The concerted action of both these repair pathways results in formation of persistent double strand breaks ultimately leading to cell death. Inactivation of either repair pathway selectively improves UV-resistance of cells expressing UmuC_Y11A.</p