DNA bending in the mycobacterial plasmid pAL5000 origin-RepB complex

Abstract

Plasmid pAL5000 represents a family of relatively newly discovered cryptic plasmids in gram-positive Actinomycetes bacteria. The replication regions of these plasmids comprise a bicistronic operon, repA-repB, encoding two replication proteins. Located upstream is a cis-acting element that functions as the origin of replication. It comprises an approximately 200-bp segment spanning two binding sites for the replication protein RepB, a low-affinity (L) site and a high-affinity (H) site separated by an approximately 40-bp spacer sequence. The trajectory of the DNA in the RepB-origin complex has been investigated, and it has been found that the origin undergoes significant bending movements upon RepB binding. RepB binding not only led to local bending effects but also caused a long-range polar curvature which affected the DNA sequences 3' to the H site. These movements appear to be essential for the in-phase alignment of the L and H sites that leads to the formation of a looped structure. A novel property of RepB unearthed in this study is its ability to form multimers. This property may be an important factor that determines the overall trajectory of the DNA in the RepB-origin complex. The results presented in this study suggest that the origins of replication of pAL5000 and related plasmids are highly flexible and that multimeric, RepB-like initiator proteins bind the origin and induce local deformations and long-range curvatures which are probably necessary for the proper functioning of the origin.

FIG. 1.

The replication region of pAL5000. (A) The minimal replication region of pAL5000 (1.8 kb) comprises a 200-bp origin and two translationally coupled open reading frames that express RepA and RepB, respectively. The core DNA sequence (90 bp; nucleotides [nt] 4531 to 4620) spanning the origin (L and H sites) is shown below. Approximately at the middle is located a conserved element (hatched box), which is present in the origins of the pAL5000 and ColE2 families of plasmids. (B to E) Concentration-dependent (0, 50, 100, 200, 400, and 800 nM; lanes 1 to 6, respectively) binding of RepB to the indicated probes. L-H (nt 4459 to 4663), H (nt 4589 to 4623), and L (nt 4531 to 4552) represent either the complete origin or the individual H and L RepB binding sites, respectively. The complexes were numbered C1 to C3. Complexes formed with the individual H and L sites were named by adding the letter H or L in parentheses. (F and G) Top-strand (F) and bottom-strand (G) DNase I footprinting using the L-H probe and RepB at a concentration of 800 nM. The A+G lane is indicated. Black bars indicate the H region, and white bars indicate the L region. − and + represent footprinting in the absence or presence of RepB, respectively.

FIG. 2.

Phasing experiments. (A) Insertions (5 or 10 bp) were made as shown, and their effect on RepB binding was assessed by performing EMSA. (B) The probe (90 bp) used for EMSA represents the minimal L-H site. RepB concentrations used were 0, 400, 600, and 800 nM, in lanes 1 to 4, 5 to 8, and 9 to 12, respectively. Complex (C1, C2, and C3) and free (F) probes are indicated. (C) Relative transformation efficiency (RTE) of pMC2-1 (5 bp) and pMC2-2 (10 bp), taking the transformation efficiency of pMC2 as unity.

FIG. 3.

Cyclization assays. A 210-bp XhoI fragment excised from pBendLH2 (see Fig. 4) which harbors the minimal L-H fragment approximately at its center was labeled at the 5′ ends with [γ-³²P]ATP and subjected to ligation using T4 DNA ligase either in the absence (lanes 1 and 5) or in the presence (lanes 2 to 4 and 6 to 8) of increasing concentrations (200, 400, and 800 nM) of RepB. Samples were either untreated (−) or treated (+) with ExoIII and analyzed on a polyacrylamide gel (5%), followed by autoradiography. The sizes of the linear multimers are indicated on the left, and the migration positions of the monomeric and multimeric circles on the right.

FIG. 4.

Circular permutation assays. The 34-bp H-site and 90-bp L-H-site fragments were cloned at the XbaI site of pBend2. Fragments for circular permutation assays were then generated by restriction digestion with MluI (M), NheI (N), XhoI (X), EcoRV (E), and BamHI (B) and labeled with [γ-³²P]ATP for performing EMSA. RepB complexes with the H site (A) or L-H site (D, E) were formed using RepB at the indicated concentrations. The naming of the complexes was done as described in the Fig. 1 legend. The sequences of presentation of the fragments were reversed in the case of the L-H site, to maintain the correct orientation of the H site. The relative positions of the H and L-H sites in the excised fragment are shown in panels B and G, respectively, with the down arrow pointing to the center of the fragment. In panel G, white, hatched, and black boxes represent L, spacer, and H regions, respectively. For analysis, R_L (μ_max/μ_min) was plotted against the position of the insert (with the midpoint taken as the reference) relative to the left end of the fragment. The plots for the indicated complexes were derived from the autoradiograms as follows: panel C represents the results for the major complex C2(H) shown in panel A; panel F represents the results for the C1 and C2 (250 and 500 μM) complexes formed with the L-H probe.

FIG. 5.

Chemical foot-printing using KMnO₄. (A and B) Autoradiograms showing KMnO₄ reactivity of the top and bottom strands, respectively, either in the absence (lanes 2) or in the presence (400 and 800 nM; lanes 3 and 4) of RepB. Lanes 1 represent A+G ladders. RepB-dependent KMnO₄-hypersensitive sites are shown by arrows in the autoradiograms (A and B) and in the origin sequence (C). The L and H sites are indicated by white and black bars.

FIG. 6.

Oligomerization of RepB. (A) SEC using standard markers and RepB (15 μM) as indicated. The x axis corresponds to the total run time (minutes). The sizes of the marker proteins are indicated at the top. The extent of multimerization (abbreviated as Mer) associated with the three peaks, I, II, and III, is indicated. (B) Western blot analysis of eluted fractions using anti-RepB serum. (C) Glutaraldehyde cross-linking of RepB. Western blot analysis of glutaraldehyde (Glu) cross-linked products using anti-RepB antibody. The M_rs of size standards are indicated on the left, and the degree of multimerization on the right. +, present; −, absent.

FIG. 7.

Model depicting RepB-origin interaction. The white, black, hatched, and dotted regions represent L, H, spacer, and flanking sequences, respectively. The loop is created in such a way that the DNA flanking the 3′ end of the H site folds back and makes secondary contacts with the H-site-bound RepB. The KMnO₄-oxidizable sites are indicated by bubbles.