DNA Pol Research Articles

Depletion of RNASEH2 Activity Leads to Accumulation of DNA Double-strand Breaks and Reduced Cellular Survivability in T Cell Leukemia

Ribonuclease H2 (RNase H2) is a member of the ribonuclease H family of enzymes involved in removal of RNA from RNA-DNA hybrids as well as ribonucleotides which get misincorporated into the genomic DNA. Recent studies have shown that RNase H2 function is also needed for successful DNA repair through NHEJ events where DNA pol µ uses ribonucleotides during the gap filling stage. Mammalian RNase H2 is composed of three subunits, RNASEH2A, RNASEH2B and RNASEH2C. There have been studies suggesting changes in expression of these genes in various cancers of breast, prostate, colon, liver, and kidney. In this study, we have investigated the functional role of RNASEH2A and RNASEH2B in leukemic T-cells, MOLT4 and Jurkat. shRNA mediated knockdown of RNASEH2A/ RNASEH2B expression led to reduced cell survival and increase in apoptotic cell population. Importantly, knockdown of RNASEH2A or RNASEH2B, led to cell cycle arrest at S phase and increased number of 53BP1 foci due to abrogation of NHEJ. Interestingly, RNASEH2A or RNASEH2B depleted cells showed significantly retarded DSB repair kinetics compared to scrambled shRNA control, when exposed to ionizing radiation suggesting that NHEJ is abrogated due to loss of RNASEH2 activity in T-ALL cells. Thus, we uncover the importance of RNase H2 function in leukemic cells and suggest that it can be targeted for cancer therapy.

Role of PPAR-γ in diabetes-induced testicular dysfunction, oxidative DNA damage and repair in leptin receptor-deficient obese type 2 diabetic mice

The testis expresses peroxisome proliferator-activated receptor-γ (PPAR-γ), but its involvement in regulating diabetes-induced testicular dysfunction and DNA damage repair is not known. Pioglitazone-induced activation of PPAR-γ for 12 weeks in db/db obese diabetic mice increases bodyweights and reduces blood glucose levels, but PPAR-γ inhibition by 2-chloro-5-nitro-N-phenylbenzamide does not alter these parameters; instead, improves testis and epididymis weights and sperm count. Neither activation nor inhibition of PPAR-γ normalizes the diabetes-induced seminiferous epithelial degeneration. The PPAR-γ activation normalizes testicular lipid peroxidation, but its inhibition reduces lipid peroxidation and oxidative DNA damage (8-oxo-dG) in diabetic mice. As a response to diabetes-induced oxidative DNA damage, the base-excision repair (BER) mechanism proteins- 8-oxoguanine DNA glycosylases (OGG1/2) and X-ray repair cross-complementing protein-1 (XRCC1) increase, whereas the redox-factor-1 (REF1), DNA polymerase (pol) δ and poly (ADP-ribose) polymerase-1 (PARP1) show a tendency to increase suggesting an attempt to repair the oxidative DNA damage. The PPAR-γ stimulation inhibits OGG2, DNA pol δ, and XRCC1 in diabetic mice testes, but PPAR-γ inhibition reduces oxidative DNA damage and normalizes BER protein levels. In conclusion, type 2 diabetes negatively affects testicular structure and function and increases oxidative DNA damage and BER protein levels due to increased DNA damage. The PPAR-γ modulation does not significantly affect the structural changes in the testis. The PPAR-γ stimulation aggravates diabetes-induced effects on testis, including oxidative DNA damage and BER proteins, but PPAR-γ inhibition marginally recovers these diabetic effects indicating the involvement of the receptor in the reproductive effects of diabetes.

In silico design of short hairpin RNA (shRNA)Molecules for DNA pol gene of Contagious Ecthyma virus (ORFV)

Contagious ecthyma is an infectious skin disease of ruminants caused by the ORF virus (ORFV) that is a member of genus Parapoxvirus of the Poxviridae family. In addition to the significant effects on lambs and human, ORFVs have been recently shown to infect other hosts. The disease causes significant economic damages to the sheep industry, so attempts to eliminate it must be taken into.RNA interference (RNAi) is an evolutionarily conserved mechanism in which the expression of homologous target genesis is suppressed by means of double-stranded RNA molecules. Since RNAi can be considered as a therapeutic method for viral gene silencing, we tend to make the most of this capability. The present study aims to design potential shRNAs to knockdown the DNA-polymerase gene coded by ORF025.A significant number of computational methods such as clustal omega website to target alignment, BLAST-NCBI to similarity search, CLC software to secondary structure prediction, BLOCK-iTRNAi Designer and WI siRNA Selection Program and Software to design of shRNA molecules and scoring have been applied for shRNA molecules designing against the ORF025-DNA pol gene of ORFV. Then three shRNA molecules were logically designed against the ORF025-DNA pol gene. In conclusion the present study provides a strong and superior approach for achieving a validated strategy to design an antiviral shRNA molecule that meets many sequence features for efficient ORFV knockdown and treatment at the mRNA level. The efficiency of these anti ORFV shRNAs need to be tested

Acyclovir-Resistant Herpes Simplex Virus 1 Keratitis: A Concerning and Emerging Clinical Challenge

To describe the clinical and virological profiles of patients with herpes simplex keratitis (HSK) caused by acyclovir-resistant (ACVR) strains of herpes simplex virus 1 (HSV-1). Multicenter retrospective case series. HSV-1 resistance to ACV was confirmed using sequencing of genes encoding HSV-1 thymidine kinase (TK) and DNA polymerase (DNA pol). Data were collected on the number of HSK episodes before and after the diagnosis of resistance, ocular findings including the type of HSK, immune status of patients, antiviral treatments, and HSV-1 genotypic resistance profiles. This study evaluated 18 HSK patients (13 male and 5 female, aged 66.8 ± 4.7 years) with ACVR HSV-1-positive ocular samples. Genotypic resistance testing was performed because of frequent recurrences despite adequate antiviral prophylaxis (AVP) (n=13, 72%), or poor response to suppressive antiviral therapy (n=5, 28%). Resistance mutations were found in the TK (n=15, 83%) or in the DNA pol gene (n=3, 17%). Prior to the diagnosis of resistance, the duration of disease was 29.8 ± 20.4 years, with more than 10 HSK recurrences in 15 patients (83%). The number of recurrences between the first episode and the diagnosis of resistance was significantly lower in immunocompromised patients (n=6, 33%) than in immunocompetent patients (n=12; 67%) (11.5 ± 4.9 vs 16.4 ± 1.9, P=.05). HSV-1 resistance to ACV must be suspected in HSK patients with recurrences despite AVP and/or in cases that respond poorly to a suppressive antiviral regimen. Immunocompromised patients and/or those with longstanding disease may be particularly at risk for developing resistance.

Beyond the Lesion: Back to High Fidelity DNA Synthesis.

High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.

Assessment of Cost-Efficient Thermocycler Prototype for Polymerase Chain Reaction and Loop-Mediated Isothermal Amplification

DNA amplification-based diagnostic is the most accurate method among others, especially during the COVID-19 pandemic. Thus, increasing the global demand for instrumentation and amplification reagents locally, hence increasing import. It is a worrying state in terms of logistics and the future domestic market. An effort for domestic production is a must. Previously a cost-efficient thermocycler prototype using Raspberry Pi and Phyton coding is constructed. Thermocycler prototype flow measurement and heat distribution have previously been tested but never put on a real test for DNA amplification diagnostic. This research aims to compare thermocycler prototypes and commercial for in two types of DNA amplification reactions, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). PCR is the most and more common method than LAMP, with the main difference of PCR require thermal cycling and LAMP operate in isothermal conditions. LAMP has a quicker reaction time and operates at a lower temperature. DNA pol with high strand displacement activity is used for LAMP, in this research Bsm pol is used for LAMP and Taq pol for PCR. Since the prototype thermocycler is designed to be as simple and inexpensive as possible for ease of manufacture and accessibility for every layer of society. Hence, its heat control and stability are not as good as a commercial thermocycler, with huge temperature fluctuation resonance from its set-point. That causes prototype incapability of performing PCR, no DNA band at 250-500 bp range in gel electrophoresis. However, the prototype is capable of performing LAMP, existing <100 bp DNA gradient band in gel electrophoresis. The prototype is also capable of performing LAMP below its protocol temperature and time separately, 62

Development of duplex real-time PCR assay based on C18L and DNA pol genes for rapid diagnosis of camel pox in dromedary camels.

Development of duplex real-time PCR assay based on C18L and DNA pol genes for rapid diagnosis of camel pox in dromedary camels.

Repair of Hypoxanthine in DNA Revealed by DNA Glycosylases and Endonucleases From Hyperthermophilic Archaea.

Since hyperthermophilic Archaea (HA) thrive in high-temperature environments, which accelerate the rates of deamination of base in DNA, their genomic stability is facing a severe challenge. Hypoxanthine (Hx) is one of the common deaminated bases in DNA. Generally, replication of Hx in DNA before repaired causes AT → GC mutation. Biochemical data have demonstrated that 3-methyladenine DNA glycosylase II (AlkA) and Family V uracil DNA glycosylase (UDG) from HA could excise Hx from DNA, thus triggering a base excision repair (BER) process for Hx repair. Besides, three endonucleases have been reported from HA: Endonuclease V (EndoV), Endonuclease Q (EndoQ), and Endonuclease NucS (EndoNucS), capable of cleaving Hx-containing DNA, thereby providing alternative pathways for Hx repair. Both EndoV and EndoQ could cleave one DNA strand with Hx, thus forming a nick and further initiating an alternative excision repair (AER) process for the follow-up repair. By comparison, EndoNucS cleaves both strands of Hx-containing DNA in a restriction endonuclease manner, thus producing a double-stranded break (DSB). This created DSB might be repaired by homologous recombination (HR) or by a combination activity of DNA polymerase (DNA pol), flap endonuclease 1 (FEN1), and DNA ligase (DNA lig). Herein, we reviewed the most recent advances in repair of Hx in DNA triggered by DNA glycosylases and endonucleases from HA, and proposed future research directions.

Susceptibility Evaluation of Clinically Isolated HSV-1 Strains to Acyclovir: A Phenotypic and Genotypic Study

Background: Mutations in herpes simplex virus Thymidine kinase (TK, UL23) and DNA polymerase (pol, UL30) genes may confer resistance to acyclovir (ACV). Phenotypic resistance must be determined along with genotypic resistance to achieve complete acyclovir susceptibility. Objectives: The present study aimed to characterize HSV-1 clinical isolates from outpatients and organ transplant recipients in terms of phenotypic ACV resistance. Moreover, genotypic resistance to ACV was assessed through sequencing the viral TK and pol genes amplified from virus-infected cell DNA. Methods: Twenty-six HSV-1 clinical isolates collected between 2016 and 2019 were examined for drug susceptibility. The samples were collected from eyes, oropharyngeal, facial, and other skin parts of immunocompetent and immunocompromised individuals. Phenotypic susceptibility was determined by using three different concentrations of ACV. The results were expressed based on the ability of ACV in reducing viral plaques by 50%. Genotyping was carried out by polymerase chain reaction and sequencing of TK and pol genes. Results: All the strains were characterized as sensitive at 0.01 and 0.05 µg.ml-1 concentrations to ACV. Seventy percent inhibition was observed at ≥ 0.1 µg.mL-1 of ACV for three isolates (two from patients who received transplants and one from an outpatient). Nine natural polymorphisms were detected in the TK gene and 31 in the Pol gene. Furthermore, four susceptible-associated mutations in the DNA pol gene were analyzed. A substitution was encoded in the conserved region of the pol Exo III motif (M553L), and nine amino acid substitutions in TK were detected. The phylogenetic analysis of partial genome sequences revealed high diversity in the TK and pol genes of HSV-1. Conclusions: A higher number of mutations were observed in patients who received transplants and underwent long-term treatment compared with outpatients. The high genetic variability of HSV-1 TK and DNA pol was not associated with phenotypic resistance.

The stabilized Pol31-Pol3 interface counteracts Pol32 ablation with differential effects on repair.

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3-Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31-Pol3 and Pol31-Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

CUT Domain Proteins in DNA Repair and Cancer.

Recent studies revealed that CUT domains function as accessory factors that accelerate DNA repair by stimulating the enzymatic activities of the base excision repair enzymes OGG1, APE1, and DNA pol β. Strikingly, the role of CUT domain proteins in DNA repair is exploited by cancer cells to facilitate their survival. Cancer cells in which the RAS pathway is activated produce an excess of reactive oxygen species (ROS) which, if not counterbalanced by increased production of antioxidants, causes sustained oxidative DNA damage and, ultimately, cell senescence. These cancer cells can adapt by increasing their capacity to repair oxidative DNA damage in part through elevated expression of CUT domain proteins such as CUX1, CUX2, or SATB1. In particular, CUX1 overexpression was shown to cooperate with RAS in the formation of mammary and lung tumors in mice. Conversely, knockdown of CUX1, CUX2, or SATB1 was found to be synthetic lethal in cancer cells exhibiting high ROS levels as a consequence of activating mutations in KRAS, HRAS, BRAF, or EGFR. Importantly, as a byproduct of their adaptation, cancer cells that overexpress CUT domain proteins exhibit increased resistance to genotoxic treatments such as ionizing radiation, temozolomide, and cisplatin.

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

AbstractDNA and RNA polymerases (Pols) are central to life, health, and biotechnology because they allow the flow of genetic information in biological systems. Importantly, Pol function and (de)regulation are linked to human diseases, notably cancer (DNA Pols) and viral infections (RNA Pols) such as COVID‐19. In addition, Pols are used in various applications such as synthesis of artificial genetic polymers and DNA amplification in molecular biology, medicine, and forensic analysis. Because of all of this, the field of Pols is an intense research area, in which computational studies contribute to elucidating experimentally inaccessible atomistic details of Pol function. In detail, Pols catalyze the replication, transcription, and repair of nucleic acids through the addition, via a nucleotidyl transfer reaction, of a nucleotide to the 3′‐end of the growing nucleic acid strand. Here, we analyze how computational methods, including force‐field‐based molecular dynamics, quantum mechanics/molecular mechanics, and free energy simulations, have advanced our understanding of Pols. We examine the complex interaction of chemical and physical events during Pol catalysis, like metal‐aided enzymatic reactions for nucleotide addition and large conformational rearrangements for substrate selection and binding. We also discuss the role of computational approaches in understanding the origin of Pol fidelity—the ability of Pols to incorporate the correct nucleotide that forms a Watson–Crick base pair with the base of the template nucleic acid strand. Finally, we explore how computations can accelerate the discovery of Pol‐targeting drugs and engineering of artificial Pols for synthetic and biotechnological applications.This article is categorized under:Structure and Mechanism &gt; Reaction Mechanisms and CatalysisStructure and Mechanism &gt; Computational Biochemistry and BiophysicsSoftware &gt; Molecular Modeling

Identification of allosteric regulation sites of Kaposis sarcoma virus DNA polymerase to discover its specific inhibitors via analysis of correlated intramolecular motions

Human herpesvirus 8 (HHV8) also known as Kaposi sarcoma herpesvirus (KSHV) is a DNA virus associated with several dangerous diseases (Castleman disease, inflammatory cytokine syndrome, Kaposi sarcoma itself, etc). The exceptional importance of DNA-dependent DNA polymerases (DNA pols) in double-strand DNA viruses' lifecycle makes them attractive targets for specific low-weight molecular inhibitors, which can be used as antiviral drugs. Among the drug discovery strategies, the search/design of allosteric inhibitors of target enzymes seems to be the most promising. As a rule, allosteric regulatory sites have individual amino acid compositions, and the selectivity of the appropriate allosteric inhibitors is much higher compared to competitive inhibitors, especially in the case of nucleotide-binding enzymes. The general structural regularities of the location of allosteric regulation sites on the protein surface remain unclear. However, it is quite natural to assume some interdependence between behavior of amino acids in active and allosteric binding site that can be revealed by correlation analysis of intramolecular motions. Thus, the aim of this work was identification of site(-s) of allosteric regulation of KSHV DNA pol for the next rational design / search for compounds - potential drugs against KSHV dependent diseases. According to the aim the following tasks are set: to calculate the molecular dynamics of KSHV DNA polymerase as well as HIV reverse transcriptase for which allosteric regulation sites are known; to evaluate a correlation between motions in active and allosteric site of HIV reverse transcriptase and on the basis of these data to determine a localization of such site(-s) on the surface of KSHV DNA polymerase; to carry out a molecular docking of several compounds with known antipolymerase activity into the identified site of allosteric regulation and estimate the appropriate binding energies. As a primary result, the correlation between the motions of amino acid residues in the active and allosteric site of HIV reverse transcriptase was revealed. The corresponding correlation coefficients are in the range of 0.55–0.62. Thus, the initial hypothesis about interdependence of the behavior of the active site and the site of allosteric regulation was confirmed. In the KSHV DNA pol was revealed a region in the range from 420 to 460 amino acid residue which motions correlate with the motions of the active site of this enzyme. The correlation coefficients are in the range of 0.52–0.57. Analysis of the molecular surface of the KSHV DNA pol revealed a site able to bind small organic compounds, which seems to be allosteric regulation site of KSHV DNA pol. As a result of molecular docking, two compounds with binding energies of -22.41 and -20.65 kJ/mol respectively were identified as potential non-competitive inhibitors of KSVH DNA pol. The identified compounds can be structural prototypes for the development of new drugs against KSHV caused pathologies. The proposed approach to the identification of allosteric regulatory sites by analysis of intramolecular correlated motions can be used for rational design/search of high selective inhibitors with a non-competitive mechanism of action for a wide range of enzymes.

Impact of Amino Acid Substitutions in Region II and Helix K of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Resistance to Foscarnet.

Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes UL30 and UL54, respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (EC50s) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA EC50s by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.

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.

Hepatitis B Virus DNA Polymerase Restrains Viral Replication Through the CREB1/HOXA Distal Transcript Antisense RNA Homeobox A13 Axis.

Long noncoding RNAs (lncRNAs) have been associated with infection and hepatitis B virus (HBV)-related diseases, though the underlying mechanisms remain unclear. We obtained HBV-HCC lncRNA profiles by deep sequencing and found HOXA distal transcript antisense RNA (HOTTIP) to be significantly up-regulated. RT-qPCR indicated that HOTTIP is highly expressed in HBV-positive hepatoma tissue and induced by HBV in vitro. Virological experiments showed that HOTTIP significantly suppresses the generation of hepatitis B viral surface antigen, hepatitis B viral e antigen and HBV replication. Homeobox A13 (HOXA13), a downstream factor of HOTTIP, was found to bind to HBV enhancer I and X promotor to repress the production of HBV pregenome RNA (pgRNA) and total RNA as well as HBV replication, suggesting that HOXA13 mediates HOTTIP-induced suppression of HBV replication. More interestingly, HBV DNA polymerase (DNA pol) binds to and stabilizes cAMP-responsive element-binding protein 1 (CREB1) mRNA to facilitate translation of the protein, which, in turn, binds to the regulatory element of HOTTIP to promote its expression. Our findings demonstrate that HBV DNA pol attenuates HBV replication through activation of the CREB1-HOTTIP-HOXA13 axis. These findings shed light on the mechanism by which HBV restrains replication to contribute to persistent infection.

Role of DNA polymerases family members in cancer: The pan-cancer perspective.

475 Background: DNA polymerases family (DNA pols) has a lengthy reported significant influence on the initiation, development, and progress of cancer. However, the pan-cancer value of whole family members was poorly done. Our study intends to demonstrate the expression pattern and clinical cancer value of DNA pols members as prognostic biomarkers and a therapeutic target of pan-cancer. Methods: Comprehensive bioinformatics analyses were done using data from TCGA and CCLE. MultiCox regression was done to select tumor prognosis-related members. Nomogram was constructed to predict the overall survival (OS) across cancer patients. Transcription factor, GO, IPA, and GSEA enrichments were done to explore regulatory mechanisms and functions. Results: A total of 22 DNA pols were identified to have a potential to diagnostic value, and 10 DNA pols have a pan-cancer prognostic value under various stages, and cancer type, among which overexpression of 6 DNA-pols (POLA2, POLD1, POLD2, POLE2, POLE4, and POLQ) was found to be significantly related to worse outcomes regarding OS, while 4 DNA-pols (POLH, POLL, POLN, and REV1) significantly related to better outcomes. A 5-DNA pols based risk score (POLQ, POLD2, POLL, POLH, and REV1) was generated by MultiCox regression with a nomogram validated an accurate predictive efficacy. MYB Proto-Oncogene Like 2 (MYBL2) was identified as transcription factors of prognostic DNA pols in pan-cancer, and IPA mimic experiment reveals inhibiting MYBL2 could be a drug target to recover and balance the dysregulated expression pattern of DNA pols in pan-cancer. GO, IPA, and GSEA enrichments revealed functions and pathways altered by DNA pols in cancer, and the results were supported by pan-cancer cell sequencing data. Conclusions: DNA pols have a pan-cancer clinical value and can work as potential prognostic biomarkers. Furthermore, MYBL2 could be a drug target for pan-cancer.

Molecular characterization of bovine foamy virus and its association with bovine leukemia virus infection in Vietnamese cattle.

The detection of bovine foamy virus (BFV) in Vietnamese cattle was performed using conventional PCR targeting pol and gag genes. Out of 243 tested samples, ten (4.1%) and eight (3.3%) samples were positive for BFV gag and pol DNA, respectively. The prevalence of bovine leukemia virus (BLV) estimated by detection of proviral DNA using nested PCR targeting env gene was 26.7% (65/243). The results of nucleotide sequence alignment and the phylogenetic analysis suggested that Vietnamese BFV strains showed high homology to isolates belonging to either European or non-European clades. There was no significant correlation between BLV and BFV. This study provides information regarding BFV infection and confirms the existence of two BFV clades among Vietnamese cattle for the first time.

Intrapatient Evolutionary Dynamics in an Individual Infected with HIV-1 CRF01_AE Who Experienced Periods of Treatment Failure.

Although previous studies have analyzed cross-level CRF01_AE viral genomic data in populations, less is known about intrapatient viral evolutionary dynamics during antiretroviral therapy (ART) failure. We longitudinally sampled plasma and peripheral blood mononuclear cells (PBMC) at different time points from one human immunodeficiency virus type 1 infected patient. The evolution of viral quasispecies was inferred from viral phylogenies. Before treatment, no drug-resistant mutations were found in this patient's plasma, and all viruses had C-C chemokine receptor type 5 (CCR5) tropism. Two months after treatment, the majority of the virus population in plasma and PBMC were drug resistant and X4-tropic. By 5 months after treatment, the viral load increased significantly, and viruses reversed tropism from X4 to R5 in plasma and PBMC. During treatment failure, the effective population of the pol DNA reservoir in PBMC remained stable, whereas the env DNA reservoir increased. The effective population of the R5 tropism virus increased more rapidly than that of the X4 tropism virus. The ratio of non-synonymous to synonymous substitutions in the env gene of R5 tropism virus (0.43) was lower than X4 tropism (0.52). However, four env positive selection sites were identified in R5 tropism viruses (HXB2: 364, 398, 399, and 400) but none were identified in X4 tropism viruses. Our data demonstrated the different intrapatient evolutionary dynamics patterns of env and pol genes in an individual who experienced periods of ART failure. Our findings also suggest the importance of the R5 tropism virus in the DNA reservoir during ART failure.

Tissue methylation and demethylation influence translesion synthesis DNA polymerases (TLS) contributing to the genesis of chromosomal abnormalities in myelodysplastic syndrome

AimsDNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of...