DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl₂, 60 mmol/L KCl, 10 mmol/L (NH₄)₂SO₄, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.
DNA-Directed DNA Polymerase/genetics* ; Escherichia coli/genetics* ; Polymerase Chain Reaction/methods* ; Temperature ; Thermococcus/genetics*

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.
DNA Primers ; genetics ; Escherichia coli ; Mutagenesis, Site-Directed ; methods ; Plasmids ; genetics ; Polymerase Chain Reaction

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to HO stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to HO-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.
DNA Damage ; drug effects ; DNA Repair ; genetics ; DNA Replication ; genetics ; DNA-Binding Proteins ; genetics ; DNA-Directed DNA Polymerase ; genetics ; Endopeptidases ; genetics ; Gene Knockout Techniques ; Humans ; Hydrogen Peroxide ; toxicity ; NEDD8 Protein ; genetics ; Oxidative Stress ; genetics ; Proliferating Cell Nuclear Antigen ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; Ubiquitin-Protein Ligases ; genetics ; Ubiquitination ; genetics ; Ultraviolet Rays

DNA Polymerase gamma ; DNA-Directed DNA Polymerase ; genetics ; Diffuse Cerebral Sclerosis of Schilder ; diagnosis ; drug therapy ; etiology ; genetics ; Heterozygote ; Humans ; Infant ; Male ; Mutation

OBJECTIVETo investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.

METHODSForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.

RESULTSCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.

CONCLUSIONCompared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

Adult ; Antiviral Agents ; pharmacology ; China ; DNA, Viral ; genetics ; metabolism ; Drug Resistance, Viral ; genetics ; Female ; HIV Infections ; drug therapy ; HIV-1 ; drug effects ; genetics ; metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Middle Aged ; Mutation ; Proviruses ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; RNA-Directed DNA Polymerase

BACKGROUND/AIMS: To investigate the association between the baseline profiles and dynamics of hepatitis B virus (HBV) DNA polymerase gene mutations and the long-term virological response of lamivudine (LAM)-adefovir (ADV) combination therapy in patients with LAM-resistant chronic hepatitis B. METHODS: Seventy-five patients who received LAM-ADV combination therapy for more than 12 months were analyzed. Restriction fragment mass polymorphism assays were used to detect and monitor the dynamics of LAM- and ADV-resistant mutations. RESULTS: The median duration of LAM-ADV combination therapy was 26 months (range, 12 to 58 months). The baseline mutation profiles, rtM204I (p=0.992), rtM204I/V (p=0.177), and rtL180M (p=0.051), were not correlated with the cumulative virological response, and the baseline HBV DNA level (p=0.032) was the only independent predictive factor for cumulative virological response. Tests for LAM- and ADV-resistant mutations were performed in 12 suboptimal responders in weeks 48 and 96. The population of rtM204 mutants persisted or increased in 8 of 12 patients, and rtA181T mutants newly emerged as a minor population in four patients until 96 weeks. Nevertheless, the viral loads progressively decreased during rescue therapy, and these dynamics did not correlate with virological response. CONCLUSIONS: The baseline profile and dynamics of LAM-resistant mutations during LAM-ADV combination therapy are not associated with a virological response.
Adenine/administration & dosage/*analogs & derivatives/therapeutic use ; Adult ; Aged ; Aged, 80 and over ; Antiviral Agents/administration & dosage/*therapeutic use ; DNA-Directed DNA Polymerase/genetics ; Drug Resistance, Viral/genetics ; Drug Therapy, Combination ; Female ; Hepatitis B virus/*genetics ; Hepatitis B, Chronic/*drug therapy/virology ; Humans ; Lamivudine/administration & dosage/*therapeutic use ; Male ; Middle Aged ; Organophosphonates/administration & dosage/*therapeutic use ; Treatment Outcome ; Viral Load/drug effects ; Young Adult

We wished to undertake molecular characterization of the reverse transcriptase (RT) gene and overlapping surface (S) gene in lamivudine-treated patients with chronic infection with the hepatitis B virus (HBV). Sequencing analyses of the HBV RT/S gene of isolates from 25 chronic hepatitis B (CHB) patients with the YMDD mutation and 30 treatment-naïve CHB patients were undertaken. In patients with the YMDD mutation, rtM2041 was the major type of mutation (20/25, 80%). rtL80I was present in most of the patients with rtM204I (14/20, 70%). rtL180M coexisted with rtM204V (5/5, 100%). Patients with the YMDD mutation had a significantly higher prevalence of mutation of the RT gene than treatment-naïve CHB patients (P < 0.05). Classical primary resistance and secondary/compensatory mutations were detected at only five sites (rtL80, rtV173, rtL180, rtM204, rtM250) in CHB patients with the YMDD mutation. The frequency of nucleos(t)ide analog resistance (NAr) mutation within the RT gene in patients with the YMDD mutation was significantly higher than that in treatment-naïve patients (P < 0.05). Amino-acid mutations within the RT gene were also associated with other types of NAr in patients with the YMDD mutation. The rate of amino-acid variants within the S gene region was significantly higher in patients with the YMDD mutation than that in treatment-naïve patients (P < 0.05). sM133L and sG145R variants were also present in patients with the YMDD mutation. These observations suggest that CHB patients with the YMDD mutation also have NAr mutations related to other NA drugs, which might lead to cross-resistance in CHB patients. Variants present in the S gene region could cause changes in the antigenicity of HBsAg, which could result in a false-negative diagnosis of HBsAg and immune in escape of the HBV.
Adolescent ; Adult ; Antigens, Surface ; genetics ; Antigens, Viral ; genetics ; DNA Mutational Analysis ; Female ; Genetic Variation ; Hepatitis B, Chronic ; drug therapy ; genetics ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; RNA-Directed DNA Polymerase ; genetics ; Young Adult

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2's Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.
Amino Acid Sequence ; Cell Cycle ; Cell Proliferation ; Chromatin Immunoprecipitation ; Cysteine ; genetics ; metabolism ; DNA Primase ; genetics ; metabolism ; DNA Replication ; DNA, Fungal ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Immunoblotting ; Immunoprecipitation ; Iron ; metabolism ; Iron-Sulfur Proteins ; metabolism ; Molecular Sequence Data ; Mutation ; genetics ; Oxidative Stress ; Protein Binding ; Saccharomyces cerevisiae ; genetics ; growth & development ; metabolism ; Sequence Homology, Amino Acid ; Sulfur ; metabolism

Avian Myeloblastosis Virus ; enzymology ; Escherichia coli ; genetics ; metabolism ; Genome, Bacterial ; MicroRNAs ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA

BACKGROUND: Most mutations in the reverse transcriptase (RT) gene of the hepatitis B virus (HBV) are related to resistance to antiviral agents. Cross-sectional studies on the mutations of this gene are rare. Thus, we analyzed the mutation patterns of RT genes and their biochemical parameters. METHODS: From 2009 to 2012, 301 blood specimens from patients with chronic hepatitis B at Daegu Catholic University Medical Center were retrospectively analyzed for the RT gene sequence of HBV, ALT, hepatitis B e antigen (HBeAg), and HBV DNA. The mutation patterns of the RT gene were compared with the biochemical parameters. RESULTS: Of the 301 patients, 100 (33.2%) had no RT gene mutations. The remaining showed the following mutation patterns: rtM204I/V (50.2%), rtL180M (39.2%), and rtA181T/V (19.6%). Combined mutations were found in 146 cases (48.5%). Of these, the combination of amino acid changes at rt180+rt204 (49.3%) was most frequently detected, followed by rt181+rt236 (11.0%) and rt173+rt180+rt204 (9.6%). In the mutated group, HBV DNA and HBeAg positive rates were significantly higher (P<0.05 for both). Phenotypic analysis showed that lamivudine resistance was most frequently detected (34.6%), followed by adefovir resistance (15.6%). Multidrug resistance was detected in 48 cases (15.9%). The adefovir-resistant group had a higher proportion of cases with HBV loads greater than 2,000 IU/mL. CONCLUSIONS: We found correlations between the mutation status of the RT domain and biochemical parameters such as HBV DNA and HBeAg positive rate. The presence of RT gene mutations could therefore be utilized to predict clinical status.
Adenine/analogs & derivatives/therapeutic use ; Antiviral Agents/therapeutic use ; DNA, Viral/analysis ; Drug Resistance, Multiple, Viral ; Drug Resistance, Viral ; Hepatitis B e Antigens/blood ; Hepatitis B virus/*enzymology/isolation & purification ; Hepatitis B, Chronic/drug therapy ; Hospitals, University ; Humans ; Lamivudine/therapeutic use ; Mutation ; Organophosphonates/therapeutic use ; Phenotype ; RNA-Directed DNA Polymerase/*genetics ; Republic of Korea ; Retrospective Studies