Changes In Polymerase Activity Research Articles

SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polβ Polymerase Activity.

In the cell, DNA polymerase β (Polβ) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polβ can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polβ variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polβ functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.

The interaction between the Nipah virus nucleocapsid protein and phosphoprotein regulates virus replication

Continued outbreaks of Henipaviruses in South Asia and Australia cause severe and lethal disease in both humans and animals. Together, with evidence of human to human transmission for Nipah virus and the lack of preventative or therapeutic measures, its threat to cause a widespread outbreak and its potential for weaponization has increased. In this study we demonstrate how overexpression of the Nipah virus nucleocapsid protein regulates viral polymerase activity and viral RNA production. By overexpressing the Nipah virus nucleocapsid protein in trans viral transcription was inhibited; however, an increase in viral genome synthesis was observed. Together, the bias of polymerase activity towards genome production led to the severe inhibition of viral progeny. We identified two domains within the nucleocapsid protein, which were each independently capable of binding the viral phosphoprotein. Evident by our data, we propose that the nucleocapsid protein’s ability to interact with the phosphoprotein of the polymerase complex causes a change in polymerase activity and subsequent deficiency in viral replication. This study not only provides insights into the dynamics of Henipavirus RNA synthesis and replication, but also provides insight into potential targets for antiviral drug development.

Identification of polymerase gene mutations that affect viral replication in H5N1 influenza viruses isolated from pigeons.

Highly pathogenic avian influenza virus H5N1 infects a wide range of host species, with a few cases of sporadic pigeon infections reported in the Middle East and Asia. However, the role of pigeons in the ecology and evolution of H5N1 viruses remains unclear. We previously reported two H5N1 virus strains, isolated from naturally infected pigeons in Egypt, that have several unique mutations in their viral polymerase genes. Here, we investigated the effect of these mutations on H5N1 polymerase activity and viral growth and identified three mutations that affected viral polymerase activity. The results showed that the PB1-V3D mutation significantly decreased polymerase activity and viral growth in both mammalian and avian cells. In contrast, the PB2-K627E and PA-K158R mutations had moderate effects: PB2-K627E decreased and PA-K158R increased polymerase activity. Structural homology modelling indicated that the PB1-V3D residue was located in the PB1 core region that interacts with PA, predicting that the PB1 mutation would produce a stronger interaction between PB1 and PA that results in decreased replication of pigeon-derived H5N1 viruses. Our results identified several unique mutations responsible for changes in polymerase activity in H5N1 virus strains isolated from infected pigeons, emphasizing the importance of avian influenza surveillance in pigeons and in studying the possible role of pigeon-derived H5N1 viruses in avian influenza virus evolution.

Mechanistic insights into the roles of three linked single-stranded template binding residues of MMLV reverse transcriptase in misincorporation and mispair extension fidelity of DNA synthesis

To obtain some insights into the structure–function relationship of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT), we modeled the catalytic state ternary complexes of this protein using the corresponding RT from human immunodeficiency virus type 1 (HIV-1) and available structures of MMLV RT. We observed that three MMLV RT single-stranded template binding residues, Y64, D114, and R116, act as a linked set through mutual interactions, including hydrogen bonding and ion-pairing. The analogous residues of HIV-1 RT have a somewhat different environment and they lack this linked phenomenon. To understand the functional implication of this linked set of MMLV RT, we performed site-directed mutagenesis at these three positions. Then the mutant enzymes were examined for their biochemical properties and nucleotide selectivity. Mutagenesis of these residues (Y64A, D114A, and R116A) resulted in enzymes with slight to modest changes in polymerase activities. The processivity of DNA synthesis correlated positively with the binding affinity of the MMLV RT variants. Lower fidelity in mutants was indicated by measurements of misincorporation and mispair extension fidelity of wild type (WT) and mutant RTs, in contrast to earlier works that indicate that mutations at the analogous positions in HIV-1 RT result in relatively higher fidelity. These data together with structural analysis suggest that this structural set may therefore be a key factor responsible for the different fidelity of these two RTs.

Triplet Entropy in H1N1 Virus

In this work we analyze triplet entropy in a set of H1N1 virus sequences. We correlated measures of entropy and guanine and cytosine concentration with chronology sample for a set of more than 30,000 sequences. This description allowed us to draw a map of the virus organizational changes throughout the 20th century. The main conclusion of our analysis was to show that some virus segments are mutating with a higher rate than others over the years and, in some cases, they are clustering sequences into new subgroups. A higher changing rate was found precisely in PA and PB1 segments, which are parts of H1N1 replication machinery. This highlights that organizational measures, as presented here, partially reflect segment dynamics and corroborates some recent experimental findings, which showed that during the last virus outbreak there has been a major change in polymerase activity. This type of approach may help to optimize treatment targets for this disease.

Role of host protein glutaredoxin 3 in the control of transcription during bacteriophage Phi2954 infection.

Bacteriophage Phi2954 contains three dsRNA genomic segments, designated L, M, and S. The RNA is located inside a core particle composed of multiple copies of a major structural protein, an RNA-dependent RNA polymerase, a hexameric NTPase, and an auxiliary protein. The core particle is covered by a shell of protein P8, and this structure is enclosed within a lipid-containing membrane. We have found that normal infection of the host Pseudomonas syringae is dependent on the action of a host protein, glutaredoxin 3 (GrxC). GrxC removes the P8 shell from the infecting particle and binds to the inner core. Removal of P8 activates the transcription of segments S and M, whereas binding of GrxC to the core particle activates the transcription of segment L. The differences in transcription behavior are due to the preference of the polymerase for G as the first base of the transcript. Transcripts of segments S and M begin with GCAA, whereas those of segment L begin with ACAA. The binding of GrxC to the particle results in changes in polymerase activity. Mutations resulting in independence of GrxC are found in the gene for protein P1, the major structural protein of the inner core particle.

Control of RNA content of developing human placenta

Human placentae obtained early in pregnancy or at full term were examined for RNA content per cell, RNA polymerase types and activities, and chromatin template availability. The RNA:DNA ratio fell from 0.7 at 15 to 20 weeks to 0.4 at 40 weeks of pregnancy. Since RNase activities were similar at both times, the reduction in RNA content was attributed not to increased degradation, but to reduced synthesis. At both stages of pregnancy, about 55 to 60 per cent of the RNA polymerase activity in isolated placental nuclei was accounted for by RNA polymerase II, as judged by suppression of activity with alpha-amanitin and by separation of the extracted polymerases on DEAE-Sephadex. The relative roles of changes in polymerase activity and template availability were measured in nuclei from 20- and 40-week placentae. Nuclei showed 20 per cent greater polymerase activity in full-term than in early placentae, but the template availability of isolated chromatin for transcription by RNA polymerase II was 70 per cent less at full term. We conclude that the reduced amount of RNA per cell in the full-term placenta is due to reduced template availability that more than offsets the slight increase in polymerase activity.

Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.

The differential effects of ions on RNA synthesis in nuclei and nucleoli isolated from neonatal and adult mouse brain

The differential effects of ionic conditions on RNA synthesis by nuclei and nucleoli isolated from neonatal and adult mouse brain was studied. Three approaches were used to discriminate between nucleoplasmic and nucleolar RNA synthesis: (1) response of RNA polymerase activity in isolated nuclei and nucleoli to various ionic conditions; (2) sensitivity of incorporation to α-amanitin, a selective inhibitor of nucleoplasmic polymerase II; and (3) use of isolated nucleoli to specifically examine nucleolar polymerase activity. The results of this investigation demonstrated that brain nuclear and nucleolar RNA synthesis respond quite differently to changes in the ionic environment. More specifically, nuclear RNA synthesis appeared to work equally well in the presence of Mg 2+ or Mn 2+ and underwent a two-fold stimulation in activity in response to high salt conditions. In contrast, nucleolar RNA synthesis clearly required the presence of Mg 2+ for significant incorporation and activity was greatly reduced in the presence of high ionic strength. The sensitivity of nuclear RNA synthesis to α-amanitin, while nucleolar activity remained refractory to the drug, suggested that the quantitative differences in polymerase activity under low and high salt conditions were also a reflection of qualitative changes in polymerase activity. Finally, nuclei and nucleoli isolated from neonatal brain tissue were found to respond in a quantitatively and qualitatively similar fashion to changes in their ionic environment as preparations from adult mice.

Proteolytic cleavage fo native DNA polymerase into two different catalytic fragments. Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage.

Treatment of native DNA polymerase from Escherichia coli with subtilisin may lead to its cleavage into a polymerase fragment and an exonuclease fragment. The cleavage has been followed by electrophoresis in polyacrylamide gel in the presence of sodium dodecylsulfate and by the change both in polymerase activity and in exonuclease activity. The electrophoretic analysis has shown that subtilisin treatment leads to the formation of two new protein bands and simultaneous disappearance of the band corresponding to the native enzyme. The cleavage of the native polymerase may be accompanied by change in the polymerase activity. The change appears to depend on the conditions of the polymerase assay. With activated calf thymus DNA the activity increases by a factor of up to 2.3 when assayed at low phosphate buffer concentration. At higher buffer concentration in the assay mixture the cleavage gives rise to a decrease in polymerase activity. With poly[d(A‐T)] · poly[d(A‐T) · the cleavage gives rise to a decrease in polymerase activity at both low and high buffer concentration. The effect of buffer concentration on the polymerase activity of the native and the cleaved enzyme showed that maximum activity for the two enzymes was obtained at different buffer concentrations in the presence of activated calf thymus DNA. In the presence of poly[d(A‐T)] · poly[d(A‐T)] maximum activity was obtained at the same buffer concentration but the activity of the cleaved enzyme was lower than that of the native at all buffer concentrations. A similar differential effect of ionic strength on the activity has been found for the exonuclease site of the native enzyme and the cleaved exonuclease fragment. This differential effect depends, however, on the molecular size of the substrate poly[d(A‐T)] · poly[d(A‐T)]. It appears, furthermore, that the exonuclease site of the cleaved exonuclease fragment has a smaller affinity towards poly[d(A‐T)] · poly[d(A‐T)] than that of the native enzyme. Finally, while the exonuclease activity of the native enzyme towards poly[d(A‐T)] · poly[d(A‐T)] is stimulated by conditions that permit simultaneous synthesis no such synthesis stimulated exonuclease activity is observed after cleavage of the native enzyme into a polymerase fragment and an exonuclease fragment.

Liver RNA polymerase activity in genetically obese and lean rats fed cholesterol

Hepatic nuclear RNA polymerase activity (C 14-ATP incorporated into RNA) as well as microsomal protein synthesis (C 14-leucine incorporated into protein) was studied in vitro in groups of genetically obese female rats of the Zucker strain before and after the introduction of a diet containing cholesterol. A similar study was carried out in normal lean rats of the same strain. The obese homozygotes were found to demonstrate a higher level of protein synthesis as well as nuclear RNA polymerase activity than did the lean homozygotes. Following the feeding of the hypercholesteremic diet for either 7 or 28 days, protein synthesis and RNA polymerase activity were shown to be lower in both groups of females. However, these changes in protein synthesis induced by this diet were relatively greater among the obese rats, whereas the concomitant changes in polymerase activity were of the same order of magnitude between genotypes. These findings have been discussed in terms of the suggested “allosteric” nature of the RNA polymerase. Furthermore, the possibility of more than one specific RNA polymerase existing in the rat liver has been speculated to explain certain of the above differences.