Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI

Abstract

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.

Structure of d(CACGCG)\cdotd(CGCGTG) in Crystals Grown in the Presence of RutheniumIIIHexammine Chloride

“…Hexammine salts are helix-stabilizing agents (Ascoli et al, 1973;Eichorn, 1982;Hingerty et al, 1982). They are especially strong inducers of Z-DNA (Winkle & Sheardy, 1990;Winkle et al, 1991;Lu et al, 1992), even in sequences that are not entirely alternating CG (Brennan et al, 1986). The absence of electron density for [Ru(NH 3 ) 6 ]Cl 3 in the present structure is an indication that the ion plays a non-speci®c role in stabilizing the structure.…”

“…It is well known that cobalt III hexammine, [Co(NH 3 ) 6 ] 3+ , induces a transition of the DNA conformation from the B-form to the Z-form (Winkle & Sheardy, 1990;Winkle et al, 1991;Lu et al, 1992) even at low concentrations (Behe & Felsenfeld, 1981). A crystal structure study of the complex of d(CGCGCG) 2 with [Co(NH 3 ) 6 ] 3+ showed that the hydrated ion bound to guanine and phosphate acceptor sites on the surface of the DNA helix .…”

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach

“…Regardless of the speciation, both will ligand sessing a single G base flanked by two A bases (COTAR1) and the oligomer possessing a CG site substitute within the 48 h reaction time as determined by AA for COTAR2 and COTAR3 as it did (COTAR4) are apparently unaffected by treatment with the cobalt complex suggesting that Co(III) for Z8 in the past. 2 We have used thermodynamic characterization does not react with either oligomer to any significant extent. However, the sequence that contains two of the thermal denaturation of DNA oligomers to study the base sequence preference of the reaction adjacent G bases (COTAR2) apparently binds Co(III) and is quite sensitive to its presence.…”

“…We have developed protocols for readdition, each oligomer possesses at least two different acting a Co(III) complex bearing a labile ligand with restriction endonuclease recognition sites: COTAR1 has a A f l II site (C/TTAAG); COTAR2 has a BamH I a DNA oligomer to yield a robust cobalt-nucleic site (G/GATCC); COTAR3 has a Hind III site (A/ acid adduct. 2 For these preliminary studies, we syn- ious DNA/cobalt ratios followed by exhaustive so-DNA Synthesizer (Applied Biosystems, Foster City, CA) dium exchange dialysis to remove any unreacted coand purified as previously described by reverse phase balt complex. Atomic absorption spectroscopic analhigh performance liquid chromatography (HPLC).…”

“…Atomic absorption spectroscopic analhigh performance liquid chromatography (HPLC). 2 After ysis of the treated oligomers indicated a gradual the second HPLC purification, the oligomers were subuptake of cobalt reaching a limiting value of one jected to exhaustive dialysis vs water and then lyophilized cobalt per four base pairs. Thermal denaturation, UV to dryness.…”