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Synthesis of chemically modified DNA
Past attempts at in vitro replication of transforming factor present in DNA have given negative or inconclusive results.(1-3) Rigid proof was lacking that template material had been excluded from the synthetic product. Even if a rigorous demonstration of net synthesis of transforming factor for a given genetic marker were forthcoming, it would still prove only that some relatively short sequence of nucleotides, sufficient for replacement of the mutant locus, had been synthesized. If enzymatic synthesis of infectious bacteriophage DNA were achieved, it would be made clear at once that relatively few, if any, mistakes had been made in replicating a DNA sequence of several thousand nucleotides.
For a few of the biosynthetic steps alternate enzymatic strategies have emerged between bacteria and vertebrates, and for such unique bacterial enzymes these present possible targets for drugs as selective antibiotics.
Protein Synthesis -Translation and Regulation
This article by Eugene Lukhtanov of ELITechGroup Molecular Diagnostics introduces the tripeptide of dihydropyrroloindole-carboxylate (CDPI3), which is a minor groove binding (MGB) moiety derived from the natural product CC-1065 and exhibits strong DNA binding properties. Synthetic oligonucleotides with covalently-attached CDPI3 moieties have enhanced DNA affinity and have improved the hybridization properties of sequence-specific DNA probes. Short CDPI3-oligonucleotides hybridize with single-stranded DNA to give more stable DNA duplexes than unmodified oligonucleotides of similar length. The author describes the general properties of CDPI3 MGB-oligonucleotide conjugates in detail, along with some specific applications:
Nucleotides form the building blocks for the synthesis of DNA and RNA, which store and transmit genetic information. They also serve as cofactors in many metabolic reactions.
Enzymatic synthesis of a DNA-templated alloy …
Phosphoramidites that allow the generation of oligonucleotides containing site-specific lesions have been vital components for studying the mechanism of DNA repair. New DNA lesions are still being discovered and the study of their biological consequences will require their site-specific incorporation into oligonucleotides. The authors conclude that the increased availability of phosphoramidites for the synthesis of lesion-containing oligonucleotides should facilitate many future discoveries in the broad area of DNA damage and repair.
The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41(mouse plasmocytoma) immunoglobulin light chain, purified to a singlepeak as shown by polyacrylamide gel electrophoresis, was used tosynthesize complementary DNA with the RNA-dependent DNA polymerase ofavian myeloblastosis virus.
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Long, Processive Enzymatic DNA Synthesis Using …
DNA is an inherently unstable molecule that can react with both endogenous (water, reactive oxygen species) and exogenous agents (UV light, environmental mutagens, antitumor agents). Errors during replication of damaged sites in DNA leads to mutations in genes and eventually cancer. More recently, it has become clear that persistent DNA lesions are associated with many additional pathologies including premature aging and neurodegeneration Fortunately, a variety of pathways exist to repair DNA to counteract this threat but mutations in DNA repair genes can lead to debilitating inherited disorders and contribute to disease predisposition.
Enzymatic Synthesis of DNA. XXIV. Synthesis of …
The bacteriophage T4 encodes ten proteins known collectively as the replisome. These proteins are responsible for the replication of the phage genome and are divided into three activities: replicase, primosomal, and Okazaki repair. T4 DNA polymerase is part of the replicase, along with the gene 45 sliding clamp, the gene 44 and 62 encoded ATP-dependent clamp loader, and the gene 32 single stranded DNA binding protein (Frankllin et al. 2001, and Mueser et al. 2010). T4 DNA polymerase is active as a monomer, but it has been suggested that dimerization is necessary for coordination of leading and lagging strand synthesis (Salinas and Benkovic 2000).
Enzymic synthesis of DNA - ResearchGate
Bacteriophage T4 DNA polymerase is also capable of an exchange (replacement) reaction. In the presence of only one dNTP the 3’ to 5’ exonuclease will degrade double stranded DNA from the 3’ hydroxyl terminus until a base is exposed that is complementary to the dNTP present. A continuous series of syntheses and exchange reactions will take place at that position.
DNA — Structure and Enzymatic Synthesis (Concluded…
The authors note that the detailed studies of the molecular mechanisms of DNA repair pathways were made possible by using site-specifically modified oligonucleotides and that the availability of phosphoramidites to synthesize oligonucleotides with DNA lesions has contributed to the field. They illustrate the article using primarily structural studies in the following examples:
Enzymatic Synthesis of DNA Complementary to …
DNA polymerase I is the predominant polymerizing enzyme found in E. coli. It contains a single disulfide bond and one sulfhydryl group (Jovin et al. 1969b). Five distinct DNA polymerases have been isolated from E. coli and have been designated I, II, III, IV, and V. DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination. DNA polymerase II also functions in editing and proofreading mainly in the lagging strand (Kim et al. 1997, Wagner and Nohmi 2000). DNA polymerase III is the main replicative enzyme. DNA polymerase IV and V have large active sites that allow for more base misincorporation, and are therefore more error-prone. They also lack proofreading-exonuclease subunits to correct misincorporations (Nohmi 2006, and Hastings et al. 2010). DNA polymerase V is present at significant levels only in SOS-induced cells and over-expression restricts DNA synthesis (Marsh and Walker 1985).
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