Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris(diphenylphenanthroline)cobalt(III).

Abstract

The chiral complex tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), A-Co(DiP)3+, binds to and, with photoactivation, cleaves left-handed DNA helices, thereby providing a unique molecular probe for local DNA conformation. We have mapped the specific left-handed sites where A-Co(DiP)3+ cleaves in the plasmids pBR322 and pLP32, which is the derivative ofpBR322 containing a Z-form d(C-G)16 insert. For pLP32, a primary cleavage is at the insert; for native pBR322, cleavage occurs at four disctte sites: 1.45, 2.3, 3.3, and 4.2 kilobase pairs. These sites correspond to segments of alternating purine-pyrimidines. Moreover, these positions map to the ends of the three distinct coding regions in pBR322: the tetracycline-resistance gene, the origin of replication, and either end of the ampicillin-resistance (.3-lactamase) gene. The locations of these left-handed segments suggest to us that Z-DNA might serve as a conformational punctuation mark to demarcate the ends of genes.Numerous spectroscopic and x-ray crystallographic studies have shown that DNA may adopt a range of conformations, from the right-handed A-and B-forms to the striking lefthanded Z-DNA helix (1-7). Regions of conformational heterogeneity along the strand, such as cruciform structures, single-stranded loops, and left-handed segments, have been detected by using DNA enzymes (8-10), and it has been suggested that local DNA conformation might play some role in regulating gene expression. Chiral metal complexes that can intercalate into the helix are particularly advantageous in probing local DNA conformation (11)(12)(13)(14). Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) complexes [Ru(DiP)"+] provide a spectroscopic probe for helix handedness; the A isomer, which does not bind B-DNA owing to steric constraints, binds avidly to Z-DNA (13). Upon photoactivation, the analogous cobalt isomers, Co(DiP)3+, furthermore cleave DNA stereospecifically, providing a sensitive assay for local regions in the Z form (14). In this report we describe the specific cleavage by A-Co(DiP)31 of discrete sites in the plasmid pLP32 (15), which contains a well-defined Z-DNA segment, and in pBR322 (16, 17). The chiral complex serves as a conformation-specific tool to map these left-handed DNA helical segments. [Co(DiP)3](tartrate)3 (10 ttM) was added to plasmid DNA (100 jkM nucleotides) in 50 mM Tris acetate buffer containing 18 mM NaCl (pH 7.0). The 20-td sample was then irradiated at 315 nm for 90 sec and ethanol-precipitated. The ethanol wash removes unreacted Co(DiP)3+ and the metal and ligand products of the reaction. After resuspension in Tris acetate buffer containing 50 mM NaCl and 10 mM MgCl2 (pH 7.0), restriction enzyme was added (EcoRI, BamHI, Ava I, or Nde I) in at least a 3-fold excess to ensure complete linearization. This was incubated at 370C for 45 min. The pH of the reaction mixture was then lowered to 5.0, and 10 mM Zn(NO3)2 was added along with 4 units of nuclease S1, and the samples were incubated for 5 min at 370C. This step causes cleavage of the...

Tris(tetramethylphenanthroline)ruthenium(II): a chiral probe that cleaves A-DNA conformations.

“…Local variations in DNA structure include bends, kinks, cruciform loops, and even left-handed Z-DNA (1)(2)(3)(4)(5). Segments of altered conformation may serve as recognition sites for the binding of regulatory proteins, and indeed the correlation of conformationally distinct sites with the borders of gene coding regions has been observed (6)(7)(8)(9).…”

“…We have focused on chiral metal complexes in developing tools to map local DNA secondary structure (10). A-tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), for example, has been shown to cleave conformationally distinct sites, such as Z-DNA, and has been used to examine altered conformations along the simian virus 40 genome (6,7,11). We have determined that tris (3,4, …”

A distinct intron-DNA structure in simian virus 40 T-antigen and adenovirus 2 E1A genes

“…On each plasmid a site within the intron insert is specifically targeted. Two other sites of specific cleavage are evident, one of which corresponds to a cruciform and another of which is located near the origin of replica ti on for the plasmid; both sites had been identified earlier in mapping studies of pBR322 with Co(DIP)J3+ (Barton & Raphael, 1985). At lower levels of irradiation of supercoiled plasmids followed by linearization but without Sl nuclease treatment, fragments Accelerated Publications Biochemistry, Vol.…”

“…However, mechanistic studies (Sitlani et al, 1992) on DNA photocleavage by Rh(phen)2phi3+, an analogue of Rh(DIP)3 3 +, on B-form DNA oligonucleotides indicate direct C3'-H atom abstraction by a photoexcited ligand radical, and this mechanism is thus far consistent with the observed cleavage by Rh(DIP)J3+, Using this cleavage methodology, Rh(DIP)J3+ has been shown to recognize unusual non-B-DNA tertiary structures such as a cruciform (Kirschenbaum et al, 1988) and a Holliday junction (Waldron et al, unpublished results). Rh(DIP)3 3 + and its structural analogue Co(DIP)J 3 + also promote cleavage on Z-DNA, but no cleavage is apparent on A-form nucleic acids or on uncoiled single-stranded sites (Kirschenbaum, 1989;Barton & Raphael, 1985). With RNA as a substrate Rh(DIPh3+ also specifically targets G-U mismatches within helical regions of RN A, while the A-RNA double helices or uncoiled regions are not cleaved (Chow & Barton, 1992b).…”

Metal–nucleic acid interactions

“…55 ,70 Perhaps even more interesting, on both natural plasmids and viral DNAs, the various sites cleaved by Co(DIPh3+, corresponding both to Z-form sites and to other locally altered non-B-conformations, coincide with functionally important regions of the genome, e.g., regulatory sites, gene termination sites, and intron-exon joints. 70,71 The altered structures recognized by the metal complexes, therefore, appear to mark biologically important sites, those presumably recognized also by cellular proteins. Cleavage studies with these metal complexes, therefore, are providing some insight also into how Nature specifically targets and accesses the sequence information encoded along the DNA polymer, sequence information encoded indirectly through local structure.…”