The effect of supercoil and temperature on the recognition of palindromic and non-palindromic regions in phi X174 replicative form DNA by S1 and Bal31.

U.R Müller,C.L Wilson

doi:10.1016/s0021-9258(18)61416-4

Abstract

The effect of supercoil and temperature on the topology of phi X174 replicative form (RF) DNA was studied using single-strand specific endonucleases S1 and Bal31 as probes for cruciform extrusion and other structural perturbations of the B-helix. Both enzymes were found to recognize specifically and reproducibly over 30 sites, most of which were cleaved by both enzymes independent of the superhelicity of the genome. A negative superhelical density exceeding 0.06 stabilized a transition in the DNA conformation that generated several new cleavage sites for Bal31. The underlying structures appeared to be only transiently stable and were lost from in vitro supercoiled DNA during brief incubation at 65 degrees C. They were generally absent from in vivo supercoiled RF DNA of equal superhelicity as a consequence of the extraction and storage procedure. Mapping of the cleavage sites suggested that they were preferentially located near the beginnings and ends of genes and that the structural basis for at least some of them was the extrusion of relatively small palindromes into the cruciform state. Insertion of a short synthetic palindromic sequence into the phi X174 genome generated a supercoil-dependent, temperature-sensitive secondary structure that was cleaved in the Bal31 but not the S1 reaction, further supporting the hypothesis that even small cruciforms with stem size of 7 or less base pairs may be transiently stable. Subjecting supercoiled RF DNA to the typical S1 reaction conditions induced a topological shift that diminished all but one of the supercoil-induced Bal31 recognition sites and promoted the formation of one major new site.

Highlights

The Effectof Supercoil and Temperatureon the Recognition of Palindromic and Non-palindromic Regions in 4x174 Replicative Form DNA by S1 and BuZ31*
Enzymes-Calf thymus topoisomerase I and nucleases S1 and Ba131 were purchased from Bethesda Research Laboratories (BRL), T4 polynucleotide kinase was from Pharmacia P-L Biochemicals or U
Most of these palindromes are relatively small, and the idea of small cruciforms being stabilized at physiological supercoil has been generally dismissed, based on some experimental evidence [21], but mostly on theoretical grounds [51].Based on the experimental findings in this report and the following consideration, we argue that cruciforms with stem sizes of 6 to 7 bp may exist at least transiently in a fraction of the DNAmolecules at physiological supercoil

Summary

RESULTS

Bacterial and Phuge Strains-Bacteriophage 4x174 strain am is referred to as wild type. Dephosphorylation and End-labeling-DNA fragments were treated with 0.2-0.4 units of calf intestinal alkaline phosphatase in either AuaI or AuaII restriction buffer. Dephosphorylated DNAs (0.5-2 pg)were incubated with 5 units of kinase and 10 pCiof [32P]ATP(approximately 3000 Ci/mmol; New England Nuclear),for 40 min at 37 "Cin restriction buffer containing 15 mM dithiothreitol and 0.1 mM spermidine. Since AuaII leaves 5"protruding single-stranded ends, thesefragments could be efficiently end-labeled with [32P]ATP.Fig. 1 shows an autoradiograph of Ba131- and S1-generated fragments separated on high resolution agarose gels. While the Bal pattern was obtained reproducibly with different DNA preparations, the S1 reaction varied, but only with regard to themajor band. This is shown in lane C, where the same S1 reaction was carried out with adifferent RF DNA preparation. With theexception of band 22, the typical S1 pattern was obtained

Since it is known that theformation of at least fairly large

Nucleotide Numbet

DISCUSSION