Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

Abstract

Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by Si nuclease cleavage. Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule.Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property. Thus molecular size is unimportant for the Sl hypersensitivity of the ColEl inverted repeat. Furthermore, a 440 bp Sau3AI, EcoRI fragment of ColEl which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits Sl cleavage similar to ColEl in the supercoiled recombinant molecule. The effect is therefore both local and transmissible. Direct competition between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated. Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising. JLNTRODUCTIONA striking feature of DNA B-form structure is its continuous symmetrical nature (1-3). However from a functional point of view it is equally apparent that discontinuities must be introduced into DNA structure in a sequencedependent manner in order to delineate functionally significant loci such as promoters, replication origins, phasing signals and other putative sites of DNA-protein interaction. Recently it has beccme apparent that DNA may be rather more polymorphic and heterologous (4-6) than has previously been considered. Many fundamental genetic processes such as RNA polymerase initiation events, require localised loss of base pairing, and thus any potential mechanism providing specific opening of the double helix is of considerable importance.I have demonstrated (7) that inverted repeats separated by short nonrepetitious sections of DM are specifically cleaved by the single strand

Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA

“…No specific sequences are seen at the nicks. The simple cleavage patterns resemble S1 nuclease cleavage patterns in inverted repeat sequences with the potential to form a pair of hairpins or a cruciform (13). Consistent with recognition of a hairpin structure, the major cleavages occur in the potential non-base-paired loop as opposed to the base-paired stem.…”

“…The sequences in the region susceptible to cleavage differ from inverted repeat sequences (potential hairpins) (13), poly purine-poly pyrimidine sequences (16)(17)(18)(19), and alternating purine-pyrimidine sequences (potential Z-DNA) (36,37) associated with S1 nuclease sites. Also, multiple, preferred cleavages over such a long stretch of DNA have not been observed using S1 nuclease (13,(16)(17)(18)(19). Our findings suggest that the altered DNA conformation detected by mung bean nuclease differs from those detected using S1 nuclease.…”

“…Lilley (13) has suggested that limited G-T, A-C pairing is permitted in potential hairpins detected under S1 nuclease conditions. When such pairing is permitted and a computer search (17) of the pBR322 sequence is performed, many additional inverted repeat sequences can be found which conform to the stem/loop sizes of the potential hairpins detected by mung bean nuclease; however, where examined at the nucleotide sequence level, cleavages were not detected at these sites (positions 2309, 2287, 3236, 3257, 4325) suggesting that limited G-T, A-C pairing are not favorable under our conditions.…”

“…In light of this possibility, the location and nature of sites cleaved by single-strand-specific endonucleases which can recognize DNA unwinding or unpairing are of considerable interest. Procaryotic supercoiled DNA (plasmid and phage) is cleaved by S1 nuclease at inverted repeat sequences but a specific relationship between the cleavage sites and genes has not been found (12)(13)(14).…”

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

“…In several isolated DNAs this enzyme preferentially cuts at the joint of short inverted repeats and this cut is prevented by relaxing the superhelical turns [17][18][19]. Direct evidence for the formation of cruciforms in supertwisted DNA was obtained by recent in vitro studies of plasmid DNAs with short natural imperfect palindromes [20,21] or longer (constructed) perfect palindromes (Refs.…”