Bacterial DNA Polymerase I

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.

The lengths of ultraviolet irradiation-induced repair resynthesis patches were measured in repair-competent extracts of Escherichia coli. Extracts containing wild-type deoxyribonucleic acid (DNA) polymerase I introduced a patch 15 to 20 nucleotides in length during repair of ColE1 plasmid DNA; extracts containing the polA5 mutant form of DNA polymerase I introduced a patch only about 5 nucleotides in length in a similar reaction. The repair patch length in the presence of either DNA polymerase corresponded to the processivity of that polymerase (the average number of nucleotides added per enzyme-DNA binding event) as determined with purified enzymes and DNA treated with a nonspecific endonuclease. The base composition of the repair patch inserted by the wild-type DNA polymerase was similar to that of the bacterial genome, whereas the patch inserted by the mutant enzyme was skewed toward greater pyrimidine incorporation. This skewing is expected, considering the predominance of pyrimidine incorporation occurring at the ultraviolet lesion and the short patch made by the mutant enzyme. Since the defect in the polA5 DNA polymerase which causes premature dissociation from DNA is reflected exactly in the repair patch length, the processive mechanism of the polymerase must be a central determinant of patch length.

The effects of Escherichia coli exonuclease I, exonuclease III, and deoxyribonucleic acid (DNA) polymerase on the biological activity of mature DNA from temperate Bacillus bacteriophage phi105 were investigated. Intact DNA loses infectivity rapidly upon exposure to exonuclease III. Although there is an overall decrease in marker rescue from exonuclease III-digested DNA, digestion preferentially affects markers at the end of the genetic map. This is taken to indicate a nonpermuted gene sequence in mature DNA. Incubation of mature DNA in the presence of exonuclease I or DNA polymerase has no effect on its biological activity. The possible structure of the ends of mature phi105 DNA is discussed. The rate of digestion of mature phi105 DNA by exonuclease III is only about 1/20 the rate of lambda DNA. Results of digestion of various DNA substrates by exonuclease III indicate that the enzyme distinguishes between different DNA terminal structures.

Mobius DNA, the paper tiger

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.

BackgroundThe use of a prefabricated spacer in two-stage revision arthroplasty remains one of the few surgery strategies for infected-joint arthroplasty treatment, despite the many unidentified microorganisms in the infected joint replacements reported in some recent studies. The aim of this prospective survey was to investigate if the sonication followed by polymerase chain reaction (PCR) can improve bacterial identification on the surfaces of prefabricated spacers and if the systemic laboratory mediators of infection and positive microbiological results can take a role of predictive factors of infection and clinical failures in 2-years follow-up.MethodsThirteen patients with prosthetic joint infection were investigated. Bacterial culture and deoxyribonucleic acid (DNA) sequencing were used to detect bacteria on the surface of prefabricated spacers removed during the second stage of revision arthroplasty. The results of pre- and intraoperative culture and DNA sequencing were compared. Minimum follow-up was 2 years.ResultsThe result of tissue cultures in second-stage revision arthroplasties revealed positive results in 15 % of patients with Coagulase-negative Staphylococci (CNS) growth. Bacterial DNA was found in over 90 % of patients with negative synovial fluid culture. Positive PCR results revealed potential pathogenic bacteria and species of human and environmental microflora with low virulence. Clinical failures at final follow-up were recorded in 2 (16.6 %) patients.ConclusionThe lack of clinical signs of infection, negative culture of preoperative joint aspirate, and intraoperative specimens do not exclude the presence of bacteria on the surfaces of spacers. The positive results of sonication and molecular tests should be interpreted as real pathogenicity factors in the light of the clinical and laboratory data, especially for patients with immunodeficiency. We confirmed our previous results that sonication followed by PCR and sequencing improved bacterial identification.

To provide some insights how herbal medicines affect deoxyribonucleic acid (DNA) synthesis in the uterus, the DNA polymerase activities (alpha and beta) in uterine samples taken from rats were measured. DNA polymerase alpha activity with respect to DNA content revealed alpha cyclic change with the highest level on proestrus, while DNA polymerase beta activity showed no significant changes during the estrous cycle. The increased period of the activity coincided with that of 17 beta-estradiol (E2) but not progesterone (P4) in the blood. Injection of 10 IU pregnant mare serum gonadotropin (PMS) on day 27 of age gradually increased DNA polymerase alpha activity in uteri, while concomitant treatment with PMS and 200 micrograms Tokishakuyakusan (TS), Keishibukuryogan (KB) or Unkeito (UT) suppressed the enzyme activity, with a most remarkable effect of KB. These results suggest that TS, KB or UT suppresses in vivo DNA polymerase alpha activity induced by PMS in the rat uterus.

The human genome contains about 50 million 5‐methylcytosine bases, most of them occurring at the CpG dinucleotide sequence. Methylated CpG dinucleotides are the targets of several of the mutations, mostly transition mutations, found in human genetic diseases and cancer.

The role of DNA damage and repair in atherosclerosis: A review

Technology is transforming rapidly. Security during data transmission is an increasingly critical and essential factor for the integrity and confidentiality of data in the financial domain, such as e-commerce transactions and bank transactions, etc. We cannot overestimate the importance of encryption/decryption of information in the digital economy. The need to strengthen and secure the digital economy is urgent. Cryptography maintains the security and integrity of data kept on computers and data communicated over the internet using encryption/decryption. A new concept in cryptography named DNA cryptography has attracted the interest of information security professionals. The DNA cryptography method hides data using a DNA sequence, with DNA encryption converting binary data into the DNA sequence. Deoxy Ribonucleic Acid (DNA) is a long polymer strand having nitrogen bases adenine (A), thymine (T), cytosine (C), and guanine (G), which play an important role in plain text encoding and decoding. DNA has high storage capacity, fast processing, and high computation capacity, and is more secure than other cryptography algorithms. DNA cryptography supports both symmetric and asymmetric cryptography. DNA cryptography can encrypt numeric values, English language and unicast. The main aim of this paper is to explain different aspects of DNA cryptography and how it works. We also compare different DNA algorithms/methods proposed in a previous paper, and implement DNA cryptography using one-time pad (OTP) and amino acid sequence using java language. OTP is used for symmetric key generation and the DNA sequence is converted to an amino acid sequence to create confusion.

Application of chimeric RNA–DNA oligonucleotides to the detection of pathogenic microorganisms using surface plasmon resonance

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.

Background: Deoxyribonucleic acid (DNA) serves as the fundamental blueprint for all life, and its integrity is paramount for cellular function and organismal health. However, the genome is constantly threatened by a multitude of endogenous and exogenous factors, including reactive oxygen species from metabolism, replication errors, ultraviolet (UV) radiation, and chemical mutagens. These agents cause various DNA lesions, from single-base modifications to double-strand breaks, which can lead to mutations if left unrepaired.Aim: This article aims to elucidate the molecular foundations of DNA damage and the sophisticated cellular repair mechanisms that maintain genomic stability, exploring the consequences from the biochemical to the clinical level.Methods: The review synthesizes knowledge on DNA damage types and the corresponding repair pathways, including base excision repair (BER) for oxidized bases, nucleotide excision repair (NER) for bulky adducts, mismatch repair (MMR) for replication errors, and homologous recombination/non-homologous end joining for double-strand breaks.Results: The article demonstrates that an imbalance between DNA damage accumulation and repair efficiency is a primary driver of disease. It uses examples like sickle cell disease (point mutation) and skin cancers (unrepaired UV damage) to illustrate how specific mutations and repair deficiencies lead to pathological outcomes.Conclusion: The continuous interplay between genotoxic stress and DNA repair capacity is a critical determinant of cellular fate. When repair pathways are compromised, the resulting genomic instability accelerates aging, promotes cancer, and causes a range of genetic disorders

T4+ exhibits increased ultraviolet sensitivity on derivatives of Escherichia coli K12 or B lacking deoxyribonucleic acid (DNA) polymerase I. However, the sensitivity of T4v is not affected by the absence of host DNA polymerase. T4x and T4y also show increased sensitivity on DNA polymerase-deficient strains, but to a lesser extent than observed with wild-type T4. When T4x or T4y, but not T4+, are plated on a double mutant lacking both DNA polymerase and the uvrA gene product, a partial suppression of the polymerase effect is observed. Host ligase appears to be able to suppress to some extent the T4y phenotype but has no effect on wild-type T4 or other T4 mutants. T4xv incubated in E. coli B or B(s-1) in the presence of chloramphenicol (50 mug/ml) shows increased resistance over directly plated irradiated phage. Increased survival under the same conditions was not observed with T4+ or other T4 mutants. The repair of X-ray-damaged T4 was investigated by examining survival curves of T4+, T4x, T4y, T4ts43, and T4ts30. The repair processes were further defined by observing the effects of plating irradiated phage on various hosts including strains lacking DNA polymerase I or polynucleotide ligase. Two classes of effects were observed. Firstly, the x and y gene products seem to be involved in a repair system utilizing host ligase. Secondly, in the absence of host DNA polymerase, phage sensitivity is increased in an unknown manner which is enhanced by the presence of host uvrA gene product.