Georgiadis, Panagiotis; (1992) Studies on the repair and conformation of DNA containing O6-alkylguanine and O4-alkylthymine. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (7MB)

Abstract

The carcinogenic N-nitroso compounds alkylate DNA. Among the different products of alkylation, O6-alkylguanine and O4-alkylthymine have attracted most of the attention since they are highly mutagenic and a correlation exists between their formation and persistence, and oncogenesis in animal model systems. Both DNA adducts in E. coli and at least O6-alkylguanine in mammalian cells are repaired by enzymes, the so-called O6-alkylguanine-DNA-alkyltransferases. Based on the HPLC separation of short, self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, or O4-methylthymine from the respective parent non-alkylated oligomers, the rate constants for their repair by the E. coli ada and ogt and the human alkyltransferases were determined. Although all alkyltransferases were able to repair O6-methylguanine, O6-ethylguanine and O4-methylthymine, the relative efficiencies were found to differ significantly. Using an immunoprecipitation assay, the rates of repair of an O6-methylguanine residue in various positions in 15 base-pair DNA duplexes were measured. The sequence of the oligomers was that of the rat H-ras sequence around codon 12 and the rates of repair were found to vary up to 25-fold depending on the sequence flanking the methylguanine. An O6-methylguanine in the second position of the GGA codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation of the selective mutation of this position observed in rat mammary tumours. The avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the adduct, with the most rapidly repaired being those most easily bound to the antibody. It is suggested that the rate of repair is a reflection of the conformation of the oligomers containing O6-methylguanine. An unusual feature of DNA which is often associated with protein-DNA interactions is DNA curvature. A characteristic of curved DNA is that it has less electrophoretic mobility than normal DNA. In order to assess if alkylated adducts in DNA induce DNA curvature or flexibility, DNA duplexes containing O4-alkylthymine or O6-methylguanine were synthesized and self-ligated to form multimers with the alkylated bases out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of DNA. All the sequences containing O4-alklylthymine migrated more slowly than expected in a non-denaturing polyacrylamide gel. In general the effect was seen when the alkylated base was out of phase or in phase with the helical repeat suggesting that the altered base-pair confers flexibility which is largely isotropic, i.e has no preferred direction, rather than anisotropic flexibility or bending. The effect of O4-methylthymine in the mobility of the oligonucleotides was much greater than that of O6-methylguanine and the effect of O4-ethylthymine slightly greater than that of O4-methylthymine. DNA duplexes containing O4-alkylT:A base-pairs were more retarded, and had lower thermal point (Tm) than DNA duplexes containing O4-alkylT:G base-pairs.

Download activity - last month

Export as JSONCSVXML

Download activity - last 12 months

Export as CSVXMLJSON

Downloads by country - last 12 months

Export as XMLCSVJSON

Archive Staff Only

View Item