Aspects Of DNA Synthesis Research Articles

Thermodynamic Insights by Microscale Thermophoresis into Translesion DNA Synthesis Catalyzed by DNA Polymerases Across a Lesion of Antitumor Platinum-Acridine Complex.

Translesion synthesis (TLS) through DNA adducts of antitumor platinum complexes has been an interesting aspect of DNA synthesis in cells treated with these metal-based drugs because of its correlation to drug sensitivity. We utilized model systems employing a DNA lesion derived from a site-specific monofunctional adduct formed by antitumor [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine) at a unique G residue. The catalytic efficiency of TLS DNA polymerases, which differ in their processivity and fidelity for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the adduct of AMD, was investigated. For a deeper understanding of the factors that control the bypass of the site-specific adducts of AMD catalyzed by DNA polymerases, we also used microscale thermophoresis (MST) to measure the thermodynamic changes associated with TLS across a single, site-specific adduct formed in DNA by AMD. The relative catalytic efficiency of the investigated DNA polymerases for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the AMD adduct, was reduced. Nevertheless, incorporation of the correct C opposite the G modified by AMD of the template strand was promoted by an increasing thermodynamic stability of the resulting duplex. The reduced relative efficiency of the investigated DNA polymerases may be a consequence of the DNA intercalation of the acridine moiety of AMD and the size of the adduct. The products of the bypass of this monofunctional lesion produced by AMD and DNA polymerases also resulted from the misincorporation of dNTPs opposite the platinated G residues. The MST analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dNTPs by DNA polymerases.

Molecular radiobiology: the state of the art.

Traditional cytotoxic agents used in cancer therapy were initially discovered based on their ability to kill rapidly dividing cells. The targets of these early-generation agents were typically one or more aspects of DNA synthesis or mitosis. Thus, dose-limiting toxicities commonly associated with these agents include GI dysfunction, immunosuppression, and other consequences of injury to normal tissues in which cells are replicating under normal physiologic conditions. Although many of these agents still play an important role in cancer therapy when given concurrently with radiation therapy, the major thrust of radiobiology research in the last two decades has focused on discovering tumor-specific traits that might be exploited for more selective targeting that would enhance the efficacy of radiotherapy with less normal tissue toxicity. These newer generation molecular targeted therapies interfere with the growth of tumor cells by inhibiting genes and their protein products that are needed specifically by the tumor for survival and expansion. These agents can be complementary to radiotherapy, a spatially targeted agent. Although there have been extraordinary technical advances in radiotherapy in recent years, we are reaching the limits of improvements that radiotherapy delivery technology can bring and need different approaches. This review will highlight promising new tumor biology-based targets and other novel strategies to reduce normal tissue injury, increase tumor control, and expand the use of radiotherapy to treat widespread metastatic disease.

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

16] Analysis of the φX174 gene a protein using in Vitro DNA replication systems

The A gene of icosahedral single-stranded DNA phages encodes a protein that is required in vivo for duplex replicative form (RF) and single-stranded circular [SS(c)] viral DNA synthesis. The gene 2 protein of the filamentous single-strand DNA phages has an analogous function, both in vivo and in vitro , during phage RF and viral DNA synthesis. Indeed, the gene A protein of icosahedral phage such as ϕ X174 may be prototypic of a variety of endonucleases in nature, associated with several aspects of DNA synthesis and metabolism including recombination and transposition. The advent of recombinant DNA technology and the development of ϕ X174-specific in vitro DNA replication systems have given rise to a powerful methodology for the study of proteins such as the gene A protein. Methods developed for the analysis of multiple activities associated with the ϕ X A protein during DNA synthesis are described in this chapter. The systems described here have additional applications. ϕ X A protein-directed leading strand DNA synthesis in vitro , from recombinant plasmid templates, is used as a probe for studying mechanisms of initiation of lagging strand DNA replication. In addition, in vitro systems similar to those described have immediate applications in the analysis of molecular mechanisms of transposition.

Effect of 2',3'-dideoxythymidine-5'-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.