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Objective To determine whether HBV DNA polymerase is associated with T-cell failure and thus mediates the immune escape of HBV-related hepatocellular carcinoma （HCC） tumor cells, and to investigate the specific molecular mechanisms. Methods Liver cancer cell lines Huh7 and HepG2 stably transfected with HBV DNA polymerase expression plasmid with Flag （Flag-HBV-P） and intercellular adhesion molecule-1（ICAM1） were co-cultured with Jurkat cells, and MTT assay, qRT-PCR, and ELISA were used to measure Jurkat cell proliferation, activation （CD69 expression), and secretion of the cytokine IFN- γ. RNA-seq was used to screen for differentially expressed immune-associated molecules between stably transfected cell lines and control cells, and mRNA half-life and protein half-life assays were used to determine the specific levels of the immune-associated molecules that were affected by HBV DNA polymerase. Related websites were used to predict the transcription factors that may bind to the promoter region of this immune-associated molecule, Western blot was used to verify the effect of transcription factors on the immune-associated molecule, and rescue experiment was used to determine whether HBV DNA polymerase affects the expression level of the immune-associated molecule through this transcription factor. The independent-samples t test was used for comparison between two groups. Results The experimental group had significant reductions in Jurkat cell proliferation, activation, and cytokine secretion compared with the control group （all P<0.01）. Compared with the control group, the experimental group （Huh7 and HepG2 cell lines） had significant reductions in the mRNA and protein expression levels of ICAM1（all P<0.01）. Website prediction identified the ICAM1 promoter and preliminarily highlighted NFKB1, RELA, and STAT3. Compared with the control group， the experimental group （Huh7 and HepG2 cell lines） had a significant reduction in the protein expression level of p65（all P<0.01）. After p65 overexpression, there was a significant increase in the protein expression level of ICAM1, and after the expression of p65 was reduced, there was a significant reduction in the protein expression level of ICAM1（all P<0.01）. In the rescue experiment， there was no significant difference in the protein expression level of ICAM1 between the control group and the experimental group after p65 overexpression （all P>0.05）. After the overexpression of ICAM1, there were no significant differences in the proliferation， activation, and cytokine secretion of Jurkat cells between the control group and the experimental group （Huh7 and HepG2 cell lines）（all P>0.05）. Conclusion HBV DNA polymerase downregulates the level of ICAM1 to mediate HCC immune escape by inhibiting the expression of p65 in NF-κB. [ABSTRACT FROM AUTHOR]

Human cancers arise from the alteration of genes involved in important pathways that mainly affect cell growth and proliferation. DNA replication and DNA damages recognition and repair are among these pathways and DNA polymerases that take part in these processes are frequently involved in cancer onset and progression. For example, damaging alterations within the proofreading domain of replicative polymerases, often reported in patients affected by colorectal cancer (CRC), are considered risk factors and drivers of carcinogenesis as they can lead to the accumulation of several mutations throughout the genome. Thus, replicative polymerases can be involved in cancer when losses of their physiological functions occur. On the contrary, reparative polymerases are often involved in cancer precisely because of their physiological role. In fact, their ability to repair and bypass DNA damages, which confers genome stability, can also counteract the effect of most anticancer drugs. In addition, the altered expression can characterise some type of cancers, which exacerbates this aspect. For example, all of the DNA polymerases involved a damage bypass mechanism, known as translesion synthesis, with the only exception of polymerase theta, are downregulated in CRC. Conversely, in pancreatic ductal adenocarcinoma (PDAC), most of these polymerase result upregulated. This suggests that different types of cancer can rely on different reparative polymerases to acquire drug resistance. Here we will examine all of the aspects that link DNA polymerases with CRC and PDAC. [ABSTRACT FROM AUTHOR]

Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme–DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome. [ABSTRACT FROM AUTHOR]

Background and Objectives: The major role of various types of DNA polymerases is genome replication. However, DNA polymerases are also necessary to establish the accuracy, efficiency of replication, repairing process, and consequently decrease in mutations induced by DNA-damaging agents. Cancer initiation is usually associated with induction of mutations in specific oncogenes or tumour suppressor genes by endogenous or exogenous genotoxic agents. Various point mutations occurring in cancer cells arise from error-generating activities of DNA polymerases. Published data show that decrease in the accuracy of DNA polymerases, without affecting their activity, could be the causative agents of tumors. It has also been reported in literature that the expression of DNA polymerases is altered in human cancers compared to normal tissue. In this review, we discuss some evidences of the role of various DNA polymerases in cancer development. The results of this study showed that lack of proper activity of DNA polymerases causes tumors through increase in the number of generated mutations in the genome. Attitude towards the function of these enzymes can result in new achievements for cancer prevention, diagnosis and treatment.

Reactive oxygen species generate 8-oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine), which induces G:C→T:A transversion mutations. The knockdowns of the protein responsible for Werner syndrome (WRN), a cancer-associated DNA helicase, and DNA polymerase (pol) λ, a WRN-interacting DNA pol, cause untargeted base-substitution mutations (action-at-a-distance mutations). To examine the consequences of the dual reductions of WRN and pol λ for the mutations caused by GO, siRNAs against both proteins were introduced into human U2OS cells. A replicable plasmid DNA with the oxidised nucleobase in a unique position of the supF gene was then introduced into the double knockdown cells. The amplified DNA recovered from the cells was used to transform a bacterial indicator strain. The mutant frequency and the subsequent sequence analysis revealed that the double knockdown additively promoted the G:C→T:A substitution at the GO position and increased the action-at-a-distance mutations to a level similar to that of the single WRN knockdown. Thus, WRN and DNA pol λ seem to suppress the targeted G:C→T:A mutation at least in part independently and reduce the untargeted mutations via an identical pathway. [ABSTRACT FROM AUTHOR]

Background ACX-362E, a novel DNA polIIIC inhibitor, is a narrow-spectrum antibacterial selectively active against certain Gram-positive bacteria, including Clostridioides difficile (MIC90 = 4 µg/mL). The objectives of this phase I study was to assess the safety, pharmacokinetics, and fecal microbiome effects of ACX-362E Methods This three-part FIH phase 1, double-blind, randomized healthy volunteer trial determined the safety profile, food effect, and systemic/stool pharmacokinetics of escalating single (150, 300, 600, and 900 mg) and multiple (300 and 450 mg) doses of oral ACX-362E vs. placebo (PBO). Fecal microbiome effects (metagenomic sequencing and qPCR) of multiple-dose ACX-362E were compared with 6 subjects receiving concomitant open-label vancomycin 125 mg four times daily. Dose escalation to each new cohort occurred following review of safety and PK data by a safety oversight committee. Results Forty-four subjects received ACX-362E (single dose = 24, multiple doses = 12, food effect = 8) and 12 PBO. Overall, ACX-362E was well tolerated at all dose levels. Adverse events were generally mild and transitory, and no moderate, severe, cumulative, or dose-limiting drug-related adverse events leading to discontinuation were observed. Mean plasma half-life was approximately 2 hours and no accumulation occurred with repeated dosing (Figure 1). Systemic exposure was less than 1 μg/mL and decreased with food. Fecal concentrations during multiple dosing exceeded the C. difficile MIC by multiples of up to ~2,500. ACX-362E had minimal effect on Bacteroidetes phylum and caused significantly less dysbiosis than vancomycin (Figure 2). Conclusion This FIH clinical trial with ACX-362E demonstrated a favorable safety profile, low systemic and high fecal concentrations, and favorable gut microbiome changes compared with vancomycin. These results shows promise for further clinical development to treat C. difficile infections. Disclosures Kevin W. Garey, MS, PharmD, Acurx (Grant/Research Support), Martin Kankam, MD, PhD, MPH, Acurx Pharmaceuticals, LLC (Research Grant or Support), Julie Mercier, BS, Acurx Pharmaceuticals, LLC (Research Grant or Support), Corinne Seng Yue, BPharm, MSc, PhD, Acurx Pharmaceuticals, LLC (Grant/Research Support), Murray Ducharme, PharmD, Acurx Pharmaceuticals, LLC (Grant/Research Support), Anne J. Gonzales-Luna, PharmD, no financial relationships or conflicts of interest, M Jahangir Alam, PhD, No financial relationships or conflicts of interest, Khurshida Begum, PhD, No financial relationships or conflicts of interest, Michael Silverman, MD, Acurx Pharmaceuticals, LLC (Consultant, Employee, Shareholder). [ABSTRACT FROM AUTHOR]

Polymerases with a proofreading function in their internal 3′ to 5′ exonuclease possess high fidelity for DNA replication both in vivo and in vitro. The obstacle facing Exo+ polymerases for single nucleotide polymorphism (SNP) detection could be bypassed by using primer-3′-termini modification. This hypothesis has been well tested using three types of modified allele specific primers with: 3′ labeling, 3′ to 5′ exonuclease resistance, and 3′ dehydroxylation. Accordingly, three new SNP assaying methods have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of Exo+ polymerases. These new mutation detection assays are widely adaptable to a variety of platforms, including multi-well plate and microarray technologies. Application of Exo+ polymerases to genetic analysis, including genotyping that is mostly relevant to pharmacogenetics, high-fidelity gene expression profiling, rare mutation detection and mutation load assay, will help to accelerate the pace of personalized medicine. In this review paper, we will first introduce three new assays that we have recently developed, and then describe a number of their applications in pharmacogenetics and in other biomedical studies. [ABSTRACT FROM AUTHOR]