RESUMO
An agarose plug method for isolating high-molecular-length DNA from mammalian tissues has been developed, including from those that are difficult, such as skin. It gives high yields of DNA that contain a minimum of single-strand breaks and is readily digested by restriction and other nucleases. The method requires only simple equipment and is readily adaptable to field or clinical studies.
Assuntos
DNA/isolamento & purificação , Pele/química , Adulto , Biópsia/métodos , Southern Blotting , DNA/química , Eletroforese em Gel de Campo Pulsado , Endopeptidase K/metabolismo , Humanos , Peso Molecular , Sefarose , Manejo de EspécimesRESUMO
Restriction enzymes, such as Eco RI, Hind III, etc., which have a potential pyrimidine dimer site in their recognition sequence, fail to cleave DNA if their recognition site is modified by the formation of pyrimidine dimers as a result of UV irradiation of DNA (J. E. Cleaver, J. Mol. Biol., 170 (1983) 305-317). We have made use of this functional property of restriction enzymes to develop a rapid and sensitive assay for DNA photolyases. UV-irradiated plasmid pBR322 DNA is only partially digested when incubated with a single site enzyme Hind III even at high concentration (20 units (micrograms DNA)-1). The amount of DNA not cleaved by Hind III is determined by agarose gel electrophoresis. The effect of UV irradiation is reversed by the photoreactivation of DNA. The decrease in the amount of Hind III-resistant DNA on treatment with photolyase gives a measure of the enzymatic activity of the photolyase preparation. The advantage of using non-radioactive DNA and the high speed and simplicity of this assay make it especially suitable for use in the purification of photolyases.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/efeitos da radiação , DNA Super-Helicoidal/metabolismo , Desoxirribodipirimidina Fotoliase/análise , Sítios de Ligação , Relação Dose-Resposta à Radiação , Resistência Microbiana a Medicamentos/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Haemophilus influenzae/genética , Cinética , Plasmídeos , Dímeros de Pirimidina/metabolismo , Transformação BacterianaRESUMO
Photoreactivation of UV-irradiated DSNA with phr A photolyase from Escherichia coli was studied in the presence of yeast RNA. Mixing of RNA with UV-irradiated DNA before its treatment with photolyase inhibited the photoreactivation of DNA. Denatured (by sonication) RNA was found to be more effective in blocking photolyase action. Agarose gel electrophoresis experiments suggest that this inhibition of photoreactivation is due to interference in the binding of photolyase with UV-irradiated DNA by yeast RNA.
