The B- to Z-DNA equilibrium in vivo is perturbed by biological processes.

Abstract

Right-handed B and left-handed Z conformations coexist in equilibrium in portions of plasmids in Escherichia coli. The equilibria are influenced by the length of the sequences that undergo the structural transitions and are perturbed by biological processes. The composite results of three types of determinations indicate a supercoil density of -0.025 in vivo. The coexistence of alternative DNA conformations in living cells implies the potential of these structures or their transitions for important functions in genetic regulatory processes.Recent studies (1) Left-handed DNA was demonstrated to exist in E. coli (1) on the basis of the observation that in vitro a target (recognition) site is not methylated by its specific methylase when the site is near (5) or in (6, 7) a left-handed Z helix. However, these enzymes do act on the same target sequences when they exist in a right-handed B structure. The gene for a temperature-sensitive EcoRI methylase (M-EcoRI) was cloned in a pACYC184 derivative to give pRW1602, and conditions were established for its temperature-dependent expression (1). pRW1602 was cotransformed with any one of 10 pBR322 derivative plasmids that contained inserts with different capabilities in vitro of forming Z or cruciform structures, depending on the lengths, orientations, and types of sequences. The excellent correlations between the ability of a sequence to inhibit methylation by M'EcoRI in vivo and in vitro, as well as its relative capacity to adopt a Z-helix in vitro, established the concept of left-handed DNA in vivo.Herein, we have employed a second assay for left-handed DNA in vivo that is based on a change in linking number for plasmids with inserts that undergo conformational transitions. Also, we have substantially extended our previous observations (1) number distribution compared to the culture with chloramphenicol was observed for any of the seven examples studied in this way. The topoisomer distributions were analyzed on 1% agarose gels in TBE buffer (0.09 M Tris/0.09 M boric acid/2.5 mM EDTA, pH 8.3) containing chloroquine (3.5-6.0 ,uM) for 40 hr at 3 V/cm.In Vitro and in Vivo M-EcoRI Studies. In vitro reactions with M-EcoRI as well as in vivo assays with the temperaturesensitive M-EcoRI were performed as described (1). RESULTSLinking Number Assay. When a B-to-Z transition occurs in vivo, the supercoil density -qo in a plasmid increases (i.e., becomes less negative) due to the relaxation process. However, to maintain a constant level of -o-,, DNA gyrase further decreases the linking difference AL to a more negative value AL', until the original -o value is reestablished (9-13). By analyzing the isolated plasmid DNA on agarose gels containing chloroquine (which reverses the B-to-Z transition), those molecules that had contained Z-DNA inside the cell should appear as bands with a more negative linking difference AL' than those that did not contain Z-DNA. In other words, the appearance of a second population of topoisomers (or a complete shift of the whole population) rela...

Formation of (dA-dT)n cruciforms in Escherichia coli cells under different environmental conditions

“…The authors assumed that (i) DNA in the cell is in the form of a plectonemic superhelix and that (ii) the torsional tension of DNA in the cell can be assessed by the torsional tension of DNA in the region between two recombining sites in a synaptic complex. Zacharias et al (42) used a, approach that is very close to ours and is based on comparison of the B-to-Z transition for the (dG-dC)n inserts of various lengths in E. coli cells with the transition in vitro estimated by two-dimensional gel electrophoresis. Unfortunately, the latter estimates were made with Tris-borate buffer.…”

Cytosine methylation enhances Z-DNA formation in vivo

“…Analysis of purified native DNA on gels containing the intercalator chloroquine revealed the distribution of linking numbers generated in vivo. Thus, the appearance of a second population of topoisomers with more-negative linking numbers or the complete shift of a topoisomer population relative to a control plasmid indicated the formation of Z-DNA in vivo (7,23).The non-B conformations detected by a combination of both in vivo assays were indeed left-handed Z-DNA, as revealed by the close agreement (i) between the extents of inhibition of methylation by M EcoRI in E. coli and the capacities of the inserted sequences to form Z helices in vitro and (ii) between the extents of relaxation measured by the in vivo linking-number assay and the results of in vitro twodimensional gel electrophoresis (14,23,25).Herein, we have analyzed the influence of cytosine methylation on the supercoil-stabilized B-Z equilibrium in vivo. The M EcoRI inhibition assay and the linking-number assay were used for DNA structural analyses inside living E. coli both in the presence and in the absence of a constitutively expressed M HhaI.…”

“…Left-handed DNA (Z-DNA) is a structural alternative to right-handed DNA (B-DNA) that has been well characterized in vitro (14,17,21). Recently, it was demonstrated that Z-DNA can exist in living Escherichia coli cells and that a given sequence can coexist in the Z form and the B form in the same cell (7,8,15,22,23). The in vivo B-Z equilibrium is influenced by active biological processes (like transcription, replication, supercoil and toroid formations, and DNAprotein interactions).…”

“…The second assay was based on the well-characterized topological changes in plasmids that have undergone a B-to-Z DNA structural transition with helix unwinding (23,25). Z-DNA formation in circular plasmids causes the relax-* Corresponding author.…”

“…The first assay was based on the observation that a recognition sequence (GAATTC) is not methylated by the corresponding bacterial methyltransferase (M EcoRI) when this site is near or inside a left-handed helix, whereas other EcoRI sites in the same plasmid molecule were completely methylated (8,23). The presence of a Z structure around the unmethylated sites was confirmed by a variety of chemical, enzymatic, and topological approaches (14,16,17,21,24 On this basis, the formation of Z-DNA inside growing bacterial cells could be analyzed by a linking-number assay.…”