Micrococcus luteus Deoxyribonucleic Acid Polymerase

152

The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly[d(GA)] showed that at low ionic strength and pH 7, 3 microM spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500 microM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurred at a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500 microM spermine. Fourth, intermolecular triplexes can also form in plasmids that contain purine.pyrimidine inserts by the addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 microM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex conformation in vivo.

“…The plasmid pTC45 contains a (TC)45 insert in the polylinker region of p913 (Pulleyblank et al, 1985). Poly[d(TC)] was prepared by depurination of the duplex (Harwood & Wells, 1970).…”

Section: Methodsmentioning

confidence: 99%

Polyamines favor DNA triplex formation at neutral pH

Hampel¹,

Crosson²,

Lee³

1991

152

“…and poly [d(Gm5C)] -poly [d(Gm5C) ] and the other synthetic polymers containing 5-methylcytosine were synthesized with Escherichia coli DNA polymerase I and dm5CTP (P-L Biochemicals) as described previously (Morgan et al, 1974;Gill et al, 1974;Evans et al, 1982). Poly(dm5C) was prepared by depurination of the duplex (Harwood & Wells, 1970). Concentrations were calculated from the absorbance measurements with extinction coefficients listed previously (Morgan et al, 1979;Wells et al, 1970) except for poly-(dG)-poly(dm5C) and DNA, where was assumed to be 6600 M-1.…”

Section: Methodsmentioning

confidence: 99%

Monoclonal antibodies specific for poly(dG).cntdot.poly(dC) and poly(dG).cntdot.poly(dm5C)

Lee¹,

Woodsworth²,

Latimer³

1984

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.

“…Poly(dT), poly(dU), poly(dC), poly(dA), poly(dA)-poly(dT), and the p(dT)x oligomers (x = 2-17) were purchased from P-L Biochemicals. The repeating-sequence pyrimidine DNAs were prepared by depurination, and poly-[d(TG)] was prepared by alkaline ceasium chloride buoyant density centrifugation from the appropriate duplex DNAs as described previously (Harwood & Wells, 1970; Morgan et al, 1974Morgan et al, , 1979Lee et al, 1979). Concentrations were calculated from absorbance measurements with extinction coefficients listed previously (Morgan et al, 1979;Lee et al, 1979) or with those supplied by the manufacturer.…”

Section: Methodsmentioning

confidence: 99%

Specificity of autoimmune monoclonal Fab fragments binding to single-stranded deoxyribonucleic acid

Lee¹,

Dombroski²,

Mosmann³

1982

Fab fragments from hybridoma HEd 10 [Lee, J. S., Lewis, J.R., Morgan, A.R., Mosmann, T.R., & Singh, B. (1981) Nucleic Acids Res. 9, 1707-1721] were prepared in large amounts by papain digestion of the immunoglobulin G (IgG) fraction from ascites fluid. Binding data were generated by a fluorescence quenching technique, and binding constants [K(0)] were estimated from Scatchard plots. The Fab fragments bound tightly to poly(dT) [K(0) = 12.7 X 10(6) M-1], and analysis of binding constants for the series p(dT)2 to p(dT)17 showed that the recognition sequence consisted of four consecutive residues. The effect of ionic strength on the interaction suggested that only two phosphates were involved. Binding constants for poly(dU), poly[d(brU)], poly[d(brC)], and poly(rU) were 1.0 X 10(6) M-1, 18.8 X 10(6) M-1, 0.5 X 10(6) M-1, and less than 0.5 X 10(6) M-1, respectively, implicating the involvement of the 3, 4, and 5 positions of the pyrimidine ring as well as the deoxyribose sugar in the recognition process. At high ionic strength (0.5 M) K(0) for whole IgG binding to poly(dT) was 75 X 10(6) M-1 compared to a value of 1.1 X 10(6) M-1 for the Fab fragment. These results may have implications for the tissue damage caused by DNA-containing immune complexes in systemic lupus erythematosus.