Active Pol Research Articles

The nucleoside analog 4'-fluorouridine suppresses the replication of multiple enteroviruses by targeting 3D polymerase.

Human enteroviruses are the major pathogens causing hand-foot-and-mouth disease in infants and young children throughout the world, and infection with enterovirus is also associated with severe complications, such as aseptic meningitis and myocarditis. However, there are no antiviral drugs available to treat enteroviruses infection at present. In this study, we found that 4'-fluorouridine (4'-FlU), a nucleoside analog with low cytotoxicity, exhibited broad-spectrum activity against infections of multiple enteroviruses with EC50 values at low micromolar levels, including coxsackievirus A10 (CV-A10), CV-A16, CV-A6, CV-A7, CV-B3, enterovirus A71 (EV-A71), EV-A89, EV-D68, and echovirus 6. With further investigation, the results indicated that 4'-FlU directly interacted with the RNA-dependent RNA polymerase of enterovirus, the 3D pol, and impaired the polymerase activity of 3D pol, hence inhibiting viral RNA synthesis and significantly suppressing viral replication. Our findings suggest that 4'-FlU could be promisingly developed as a broad-spectrum direct-acting antiviral agent for anti-enteroviruses therapy.

Lipid peroxidation and the antioxidant system of the lungs during hyperbaric oxidation and its aftereffect: prospective study

OBJECTIVE. To study the state of lipid peroxidation (LP) and the antioxidant system in the lungs of a healthy organism with single and repeated use of HBO in the therapeutic regime and the dynamics of the detected changes in the posthyperoxic period.
 MATERIALS AND METHODS. The experiments were conducted on a 100 mongrel white rats (males) weighing 180-220 g. Hyperbaric oxygenation (HBO) was performed in a therapeutic “soft” mode (2 ata, 50 min), 1 session per day. The studies were carried out immediately after the first and fifth sessions of HBO, as well as on the 5th day after a single application of HBO and on the 5th knock after the end of the five-day course of HBO. The content of extraerythrocytic hemoglobin (EEG), malondialdehyde (MDA), urea, uric acid (UC), activity of superoxide dismutase (SOD) and catalase were studied in the lung tissue.
 RESULTS. A five-day course of HBO does not lead to the accumulation of EEG in the lung tissue and causes an increase in the concentration of MDA in it only after the 5th session of HBO, whereas on the 5th day of the posthyperoxic period (PHP) it is within the normal range. Adapting to the selected HBO regimen, the lung tissue responds by increasing the activity of SOD and catalase, which normalize with an increase in the number of sessions to five. Unlike SOD, the increase in catalase activity on the 5th day of PHP depends on the number of HBO sessions. If the HBO-determined accumulation of urea by the lung tissue persists on the 5th day of the post-peroxic period, then the accumulation of UC is only with hyperoxic exposure.
 DISCUSSION. The changes we have identified are a manifestation of the natural reaction of the LP and the antioxidant system of the lungs of a healthy organism to an over-saturation with oxygen carried out in a therapeutic “soft” mode of 2 ata, 50 min.
 CONCLUSION. A five-day course of HBO in a therapeutic “soft” mode (2 ata, 50 min, 1 session per day) does not cause uncontrolled activation of POL and depletion of the antioxidant system in the lung tissue.

DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation.

DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication.

Manganese-independent Reverse Transcriptase Activity of Tth DNA Polymerase with Two Amino Acid Substitutions.

The DNA polymerase of Thermus thermophilus (Tth pol) presents reverse transcriptase activity with Mn2+, and can be used for one-step RT-qPCR. However, Mn2+ would reduce amplification fidelity and cause nonspecific products. Eliminating the Mn2+ dependence of the reverse transcriptase activity of Tth pol by point mutations. We constructed three variants I640F, I709K, and I640F/I709K, and measured their DNA polymerase and reverse transcriptase activities without Mn2+. Their enzymatic characteristics and PCR inhibitor resistance were also tested. Finally, these variants were applied in one-step RT-qPCR. All three variants presented reverse transcriptase activity with Mg2+ only and increased DNA polymerase activity. The variants, except I709K, showed no significant difference in thermostability, optimal pH, optimal NaCl concentration, storage stability and PCR inhibitor resistance compared to the wild type. Variant I640F/I709K had good performance in one-step RT-qPCR with Mg2+ only, whereas both variants with single substitution exhibited nonspecific amplification. We successfully constructed three Tth pol variants possessing Mn2+ independent reverse transcriptase activity. The variant I640F/I709K was suitable for one-step RT-qPCR because of its good performance.

Polygalaxanthone III downregulates inflammation in the lipopolysaccharide-stimulated RAW264.7 macrophages: A quantibody array analysis

Polygala japonica Houtt. (PJ), a member of the Polygala L. family that is suggested to exhibit detoxification properties in traditional Chinese medicine, is often used to treat upper respiratory tract infections. The anti-inflammatory effects of four main components of PJ (POL, PS-XLIX, PS-E, and PS-F) were examined using the LPS(0.3 μg·mL−1)-stimulated RAW264.7 macrophage model. The levels of NO, ROS, and iNOS were examined to analyze the anti-inflammatory activity of POL. Additionally, the levels of extracellular inflammation-related cytokines and chemokines were measured using quantibody array. The KEGG pathway analysis was performed to examine the anti-inflammatory mechanism of POL. The levels of NO in the POL-pretreated group were significantly downregulated when compared with those in the PS-E-pretreated, PS-F-pretreated, and PS-XLIX-pretreated groups. POL significantly inhibited the changes of iNOS, ROS, and inflammatory factors caused by LPS stimulation (p < 0.001). The expression levels of IL21 and GM-CSF were examined using qPCR, while those of JAK-STAT signaling pathway-related proteins in the LPS-stimulated RAW264.7 macrophages were analyzed using western blotting. POL significantly downregulated the expression of IL-21 and GM-CSF. The anti-inflammatory mechanism of POL is mediated through the JAK-STAT pathway. Thus, this study demonstrated that POL is an anti-inflammatory component of PJ and elucidated its mechanism.

EFFECTIVENESS OF MICROELEMENTS IN THE FORMATION OF MECHANISMS OF PROTECTION OF THE BODY UNDER THE INFLUENCE OF ENVIRONMENTAL STRESS FACTORS

Analysis of literature data and the results of our own research show that even with intensive rearing of animals with the use of balanced feeding and keeping them in accordance with the technology - it is almost impossible to avoid stress. And especially, excessive man-made load on agroecosystems also has a negative impact on animal life.&#x0D; Factors that cause a decrease in immune status and the emergence of immune pathology in animals include: industrial technology of animal husbandry, chemicalization in crop and livestock production, man-made pressure, dietary imbalance in nutrients and biologically active substances. This increases the impact on the body of heavy metals, which displace nutrients from body tissues, in particular trace elements and replace them in metabolic processes, which is a potential prerequisite for the development of oxidative stress. The mechanism of development of stress reaction of an organism is closely connected with activity of POL (lipid peroxidation) and depression of antioxidant potential. Under these conditions, the ability of the organism to mobilize protective and adaptive capabilities in response to the action of negative factors becomes especially important. Keeping productive animals in adverse conditions, unbalanced feeding are components of immobilization stress, which reduces their productivity, affects reproductive function, metabolic and functional disorders, reducing nonspecific and specific resistance of the organism (Fedoruk &amp; Kravtsiv, 2003; Velychko, 2008; Velychko, 2011).&#x0D; Under conditions of man-caused load on the environment, respectively, and animals – it is promising to develop effective methods for regulating the activity of the enzymatic system of antioxidant protection with the help of biologically active substances, in particular trace elements. The results of research confirm that this has a positive effect on the formation of productive and adaptive properties of animals.&#x0D; The system of antioxidant protection is a necessary part of non-specific reactions of the organism, a component of the processes of its adaptation to environmental conditions, a component of normal life, a factor in maintaining homeostasis. Widespread immunodeficiency and elucidation of the main links of their pathogenesis have exacerbated the problem of regulating disorders of the immune system. Therefore, knowledge of the patterns of formation of the immune status of animals, especially in the early postnatal period, is important in the development of methods for the correction of immunodeficiency, antioxidant protection.

Differential impact of various substitutions at codon 715 in region II of HSV-1 and HCMV DNA polymerases

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

Features of the system of antioxidant protection and lipid peroxidation in microangiopathies against type 2 diabetes mellitus

The article presents the results of an experimental study of the AOР and LPО system in animals simulated complications of the microcirculatory tract (diabetic retinopathy, diabetic nephropathy) on the background of type 2 diabetes. It was found that in the group of animals with type 2 diabetes increased the level of DC, TBCproducts and MDA; catalase and FG levels decreased. In animals with simulated DN, AOP disorders were more pronounced compared to animals with DR. This trend can be explained by the fact that in addition to hyperglycemia in these animals, the excessive formation of free radicals contributes to metabolic shifts in ischemic areas of tissues and organs. Disturbance of balance in AOP and LPO in type 2 diabetes triggers a pathogenetic cascade of development of complications of the microcirculatory tract and is accompanied by a tendency to further increase the generation of reactive oxygen species and activation of POL in microangiopathies.

The Involvement of Mitophagy in the Prevention of UV-B-Induced Damage in Human Epidermal Keratinocytes

Human skin is influenced by ultraviolet-B (UV-B) irradiation. UV-B induces DNA damage and mitochondrial damage in epidermal keratinocytes； then accumulation of excessive DNA damage and damaged mitochondria finally leads to apoptosis. Recently, we investigated effective means to repair or to eliminate fatal damage leading to excessive apoptosis in UV-B-exposed normal human epidermal keratinocytes (NHEKs) . Concerning repair of DNA damage, we previously established a method for measuring activity of cellular DNA polymerase (pol) which synthesizes DNA strands in the repair system in NHEKs, and found that Rhodomyrtus tomentosa fruit extract, which has properties to protect epidermal keratinocytes from UV-B-induced apoptosis, enhanced activity of pol in UV-B-irradiated NHEKs. With regard to repair and elimination of mitochondrial damage, we focused on mitophagy, which is the cellular function to remove dysfunctional mitochondria by autophagy. In the present study, we detected mitophagy and evaluated mitochondrial reductase activity providing an indication of cell viability in UV-B-irradiated NHEKs. As a result of evaluating the influence of UV-B irradiation on mitophagy in NHEKs, we observed that mitophagy became weak after irradiation with 40 mJ/cm2 UV-B. In addition, mitochondrial reductase activity also decreased at 24 h after irradiation. Following these results, we also found that Rhodomyrtus tomentosa fruit extract suppressed the UV-B-induced decrease in mitophagy and in mitochondrial reductase activity. Furthermore, we investigated influence of UV-B irradiation and effect of the extract on mitochondrial membrane potential in NHEKs to elucidate the mechanisms of modulating mitophagy. The results showed that the extract retained normal function of mitophagy by decreasing mitochondrial membrane potential in advance. In conclusion, it was suggested that the enhancement of inherent cellular DNA repair properties and the efficient elimination of the mitochondrial damage are essential for protecting the epidermal keratinocytes from UV-B-induced excessive apoptosis.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Rpb5, a subunit shared by eukaryotic RNA polymerases, cooperates with prefoldin-like Bud27/URI

AbstractRpb5 is one of the five common subunits to all eukaryotic RNA polymerases, which is conserved in archaea, but not in bacteria. Among these common subunits, it is the only one that is not interchangeable between yeasts and humans, and accounts for the functional incompatibility of yeast and human subunits. Rpb5 has been proposed to contribute to the gene-specific activation of RNA pol II, notably during the infectious cycle of the hepatitis B virus, and also to participate in general transcription mediated by all eukaryotic RNA pol. The structural analysis of Rpb5 and its interaction with different transcription factors, regulators and DNA, accounts for Rpb5 being necessary to maintain the correct conformation of the shelf module of RNA pol II, which favors the proper organization of the transcription bubble and the clamp closure of the enzyme.In this work we provide details about subunit Rpb5's structure, conservation and the role it plays in transcription regulation by analyzing the different interactions with several factors, as well as its participation in the assembly of the three RNA pols, in cooperation with prefoldin-like Bud27/URI.

Rpb5, a subunit shared by eukaryotic RNA polymerases, cooperates with prefoldin-like Bud27/URI.

Rpb5 is one of the five common subunits to all eukaryotic RNA polymerases, which is conserved in archaea, but not in bacteria. Among these common subunits, it is the only one that is not interchangeable between yeasts and humans, and accounts for the functional incompatibility of yeast and human subunits. Rpb5 has been proposed to contribute to the gene-specific activation of RNA pol II, notably during the infectious cycle of the hepatitis B virus, and also to participate in general transcription mediated by all eukaryotic RNA pol. The structural analysis of Rpb5 and its interaction with different transcription factors, regulators and DNA, accounts for Rpb5 being necessary to maintain the correct conformation of the shelf module of RNA pol II, which favors the proper organization of the transcription bubble and the clamp closure of the enzyme.In this work we provide details about subunit Rpb5's structure, conservation and the role it plays in transcription regulation by analyzing the different interactions with several factors, as well as its participation in the assembly of the three RNA pols, in cooperation with prefoldin-like Bud27/URI.

The 5′ Nuclease Domain of DNA Polymerase I Mediates a Novel DNA Transfer Pathway during Proofreading

The high fidelity of many DNA polymerases is due, in part, to their ability to remove erroneously incorporated nucleotides at the terminus of the growing primer strand. This “proofreading” activity is catalyzed by the 3′-5′ exonuclease (exo) domain in E. coli DNA polymerase I (Pol I). Structural studies have shown that this domain is spatially separated from the 5′-3′ polymerase domain (pol), which is responsible for incorporation of dNTPs by template-directed polymerization. Pol I also contains another spatially distinct domain, the 5′ nuclease (nuc) domain, which is connected to the enzyme core by a flexible amino acid linker. This domain is involved in cleavage of downstream flaps formed during Okazaki fragment maturation and base excision repair. Using a single-molecule FRET system, we resolved three distinct conformations of Pol I-DNA complexes. Experiments with Pol I mutants established that these states correspond to DNA bound at the pol, exo, or nuc domains. Notably, the DNA substrate can transfer among all three domains without dissociation from the enzyme. We determined rate constants for site switching of model DNA substrates containing a correctly paired primer terminus, a terminal mismatch or an internal mismatch. The presence of mismatches accelerated transfer of DNA from the pol domain to the exo domain, as expected during proofreading. Surprisingly, the mismatches also accelerated transfer of DNA along the pol to nuc to exo domain pathway. These observations reveal the existence of a second intramolecular proofreading pathway, in which mismatched DNA first transfers from the pol domain to the nuc domain and then to the exo domain. Our results suggest an unexpected role for the 5′ nuclease domain in the proofreading activity of Pol I.

The human papillomavirus DNA helicase E1 binds, stimulates, and confers processivity to cellular DNA polymerase epsilon.

The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ’s replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1’s helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase.

N2‐Substituted 2′‐Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

AbstractN2‐Alkyl‐2′‐deoxyguanosine triphosphate (N2‐alkyl‐dGTP) derivatives with methyl, butyl, benzyl, or 4‐ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N2‐Benzyl‐dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N2‐4‐ethynylbenzyl‐dG with wild‐type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)‐FAM‐azide demonstrated that only cells containing pol κ were able to incorporate N2‐4‐ethynylbenzyl‐dG into the nucleus. This is the first instance of a Y‐family‐polymerase‐specific dNTP, and this method could be used to probe the activity of pol κ in vivo.

N2 -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ.

N2 -Alkyl-2'-deoxyguanosine triphosphate (N2 -alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N2 -Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N2 -4-ethynylbenzyl-dG with wild-type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)-FAM-azide demonstrated that only cells containing pol κ were able to incorporate N2 -4-ethynylbenzyl-dG into the nucleus. This is the first instance of a Y-family-polymerase-specific dNTP, and this method could be used to probe the activity of pol κ in vivo.

DNA Polymerase ν Rapidly Bypasses O6-Methyl-dG but Not O6-[4-(3-Pyridyl)-4-oxobutyl-dG and O2-Alkyl-dTs.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O6-methyl-2'-deoxyguanosine (O6-Me-dG), O6-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O6-POB-dG), O2-methylthymidine (O2-Me-dT), and O2-POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O6-Me-dG is a good substrate for pol ν, while O6-POB-dG and the O2-alkyl-dT adducts are poor substrates for pol ν. The reactivity with O6-Me-dG is high with a preference for dCTP > dGTP > dATP > dTTP. The catalytic activity of pol ν toward O6-Me-dG is high and can potentially be involved in its bypass in vivo. In contrast, pol ν is unlikely to bypass O6-POB-dG or the O2-alkyl-dTs in vivo.

Y-box-binding protein 1 as a non-canonical factor of base excision repair

Base excision repair (BER) is a flagship DNA repair system responsible for maintaining genome integrity. Apart from basal enzymes, this system involves several accessory factors essential for coordination and regulation of DNA processing during substrate channeling. Y-box-binding protein 1 (YB-1), a multifunctional factor that can interact with DNA, RNA, poly(ADP-ribose) and plenty of proteins including DNA repair enzymes, is increasingly considered as a non-canonical protein of BER. Here we provide quantitative characterization of YB-1 physical interactions with key BER factors such as PARP1, PARP2, APE1, NEIL1 and pol β and comparison of the full-length YB-1 and its C-terminally truncated nuclear form in regard to their binding affinities for BER proteins. Data on functional interactions reveal strong stimulation of PARP1 autopoly(ADP-ribosyl)ation and inhibition of poly(ADP-ribose) degradation by PARG in the presence of YB-1. Moreover, YB-1 is shown to stimulate AP lyase activity of NEIL1 and to inhibit dRP lyase activity of pol β on model DNA duplex structure. We also demonstrate for the first time YB-1 poly(ADP-ribosyl)ation in the presence of RNA.

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability.

DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.