Properties of DNase I digestion of the deoxyoligonucleotide: 5''d(ATCGTACGAT)2(3'').

Deoxyribonuclease I digestion of the deoxyoligodecamer 5'd(ATCGTACGAT)2(3') has been examined in detail to study the kinetic and structural properties of this enzyme substrate system in solution. In addition, these studies have defined, in general, those DNase I conditions to be used in future drug-DNA footprinting experiments. Special attention has been taken of those properties of DNase I that are critical for quantitation of ligand binding to small DNA fragments, and that aid in designing oligomers to be used in footprinting experiments. Enzyme activity was observed at all phosphodiester bonds in the decamer studied with varying affinity, except for the first four bonds at the 5' end of the oligomer. The DNA substrate concentration is always in excess, in order to achieve conditions of no more than one DNase I cleavage per DNA molecule. Reactions were controlled so that 65% or more of the initial amount of decamer substrate remained after DNase I digestion. It was observed that the rate of enzyme reactivity decreases with digestion time and is sensitive to the experimental conditions.     

Visualization of an AAF induced frameshift mutation: molecular views of base displacement in B-DNA from minimized potential energy calculations.

Energy minimized structures of base displacement in an AAF modified B-DNA dodecamer are presented. A rational search strategy, beginning with a global search of the conformation space of the modified deoxydinucleoside monophosphate, together with model building by computer graphics, has been employed. A number of different minimum energy conformations have been located which reveal base displaced structures. These show fluorene interstrand stacking, fluorene inter- and intrastrand stacking, and non-stacked fluorene situated in the denatured bulge. The local helix axis is bent to various extents in the different forms, and one or two base pairs are fully denatured. One structure of special interest offers a molecular view that suggests how AAF can induce the -2 deletion mutation observed in AAF modified E. coli.     

Efficient methods for attachment of thiol specific probes to the 3''-ends of synthetic oligodeoxyribonucleotides.

Methodology is described for the synthesis of DNA oligomers containing a free 3'-thiol group which can be selectively crosslinked with a wide variety of probes. This chemistry is compatible with both phosphotriester and phosphoramidite solid phase chemistry. Moreover, the sulphydryl group is introduced into the 3'-nucleoside solid support linkage prior to oligonucleotide synthesis. Consequently, no additional coupling steps are required after oligonucleotide synthesis, and isolation of the 3'-thiol oligonucleotide requires only one additional deprotection step. Cross-linking of the thiol-containing oligonucleotide to a fluorescent probe was carried out with high selectivity, in high yield, and under mild conditions.     

Purification and amino-terminal amino acid sequence of an apurinic/apyrimidinic endonuclease from calf thymus.

An apurinic/apyrimidinic (AP) endonuclease (E.C.3.1.25.2) has been purified 1100 fold to apparent homogeneity from calf thymus by a series of ion exchange, gel filtration and hydrophobic interaction chromatographies. The purified AP endonuclease is a monomeric protein with an apparent molecular weight on SDS-PAGE of 37,000. On gel filtration the protein behaves as a protein of apparent molecular weight 40,000. DNA cleavage by this AP endonuclease is dependent on the presence of AP sites in the DNA. DNA cleavage requires the divalent cation Mg2+ and has a broad pH optimum of 7.5-9.0. Maximal rates of catalysis occur at NaCl or KCl concentrations of 25-50 mM. The amino acid composition and the amino-terminal amino acid sequence for this AP endonuclease are presented. Comparison of the properties of this AP endonuclease purified from calf thymus with the reported properties of the human AP endonuclease purified from HeLa cells or placenta indicate that the properties of such an AP endonuclease are highly conserved in these two mammalian species.     

Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.     

2D-NMR studies of the unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG).

The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG). NOE data provide evidence that both duplexes form stable right-helical duplexes with an anti-conformation on the glycosyl linkages and a Watson-Crick pairing. NOESY and COSY spectra allowed us to determine that alpha and beta deoxyriboses adopt a 3' -exo conformation.