Linear Streptomyces plasmids form superhelical circles through interactions between their terminal proteins

Abstract

Linear chromosomes and linear plasmids of Streptomyces possess covalently bound terminal proteins (TPs) at the 5' ends of their telomeres. These TPs are proposed to act as primers for DNA synthesis that patches the single-stranded gaps at the 3' ends during replication. Most ('archetypal') Streptomyces TPs (designated Tpg) are highly conserved in size and sequence. In addition, there are a number of atypical TPs with heterologous sequences and sizes, one of which is Tpc that caps SCP1 plasmid of Streptomyces coelicolor. Interactions between the TPs on the linear Streptomyces replicons have been suggested by electrophoretic behaviors of TP-capped DNA and circular genetic maps of Streptomyces chromosomes. Using chemical cross-linking, we demonstrated intramolecular and intermolecular interactions in vivo between Tpgs, between Tpcs and between Tpg and Tpc. Interactions between the chromosomal and plasmid telomeres were also detected in vivo. The intramolecular telomere interactions produced negative superhelicity in the linear DNA, which was relaxed by topoisomerase I. Such intramolecular association between the TPs poses a post-replicational complication in the formation of a pseudo-dimeric structure that requires resolution by exchanging TPs or DNA.

Figure 1.

In vivo cross-linking of telomeres. Mycelium from liquid cultures was treated with DSS or DSG. Total DNA was isolated, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized to labeled plasmid DNA. (A) pLUS891L. The physical map of the plasmid DNA is shown above. The sizes of the restriction fragments are indicated (in kb). tsr, thiostrepton resistance gene; ARS, autonomously replicating sequence of pSLA2; filled arrows, SCP1 telomeres; filled circles, Tpc proteins. The DNA linked by the cross-linker is indicated by an asterisk. ‘M’, DNA size markers. The size of the DNA fragments is depicted on the left (in kb). A set of samples was treated with proteinase K (‘+’) before electrophoresis (right panel). (B) pLUS892L. The symbols and analyses are as in (A).

Figure 2.

In vitro cross-linking of telomeres. (A) Without GnHCl treatment. Total DNA of S. lividans 3200/pLUS891L was treated with DSS or DSG, digested with BclI (Bc) or BglII (Bg), fractionated by gel electrophoresis and hybridized with pLUS891L DNA probe. (B) With GnHCl treatment. Genomic DNA of S. lividans 3200/pLUS892L was isolated by GnHCl–CsCl gradient centrifugation and treated with DSS or DSG. The treated DNA was digested with BclI or MluI, fractionated by gel electrophoresis and hybridized to the SCP1 terminal DNA probe. The physical map of pLUS892L is shown above with the BclI and MluI (Ml) sites and fragments sizes indicated. In the control experiment (right panel), the samples were treated with proteinase K before electrophoresis. The cross-linked DNA is indicated by an asterisk.

Figure 3.

Specificity of telomere interactions analyzed by the 3C procedure. (A) Physical map of pLUS892L and the predicted cross-linked, ligated products. On pLUS892L (top), the positions of the L and R primers and their distances to the nearest MluI (‘Ml’) sites are indicated (in kb). On the expected intramolecular (middle) and intermolecular (bottom) products, the expected PCR products and their sizes (in kb) are indicated. Cross-linking is depicted by the dashed arcs. The other symbols are as in Figure 1. (B) Detection of intermolecular and intramolecular cross-linking. S. lividans 3200/pLUS892L mycelium was treated with DSS, and lysed by protoplasting and osmotic shock. DNA in the lysate was digested with MluI, and ligated at two different concentrations (top panel, 20-µl ligation reaction; bottom panel, 1-ml ligation reaction) followed by LM-PCR using the R, L or both primers (‘R + L’) in the absence (‘0%’) or presence (‘10%’) of DMSO. The PCR products were analyzed by gel electrophoresis. The sizes of the PCR products are indicated (in kb). (C) Detection of intramolecular cross-linking after GnHCl centrifugation. Cross-linking and detection were performed as in B except for an additional GnHCl–CsCl gradient centrifugation step (right half) DNA before cross-linking. The volume of the ligation reaction was 1 ml.

Figure 4.

Interactions between chromosomal telomeres and between a chromosomal and plasmid telomere. (A) Top, physical map of pLUS971 is shown above. The open arrows and open circles represent the telomeres and TPs of the S. lividans chromosome, respectively. The distances between the primers and the nearest MluI (‘Ml’) sites are indicated (in kb). Middle and bottom, 3C analysis as described in Figure 3. (B) Top, physical map of pLUS890L. The filled arrow and filled circle represent the SCP1 telomere and terminal protein, respectively. Middle and bottom, 3C analysis as described in Figure 3.

Figure 5.

Interactions between the telomeres of the S. coelicolor chromosome and the SCP1 plasmid. (A) Shown from the top are the physical maps of the termini of the S. coelicolor chromosome and SCP1 with the distances between the primers (a and b, respectively) and the nearest BamHI (‘Ba’) sites indicated (in kb), and the expected products of ligation between the telomeres of the chromosome and SCP1, between the chromosomal telomeres and between the SCP1 telomeres. The other symbols are as in Figures 3 and 4. (B) 3C analysis as described in Figure 3.

Figure 6.

Superhelical structures formed by linear plasmid DNA. (A) S. lividans 3200/pLUS892L mycelium was osmotically lysed. The cell lysate was treated with E. coli Topoisomerase I (‘Topo I’), fractionated by gel electrophoresis and hybridized to the pLUS892L DNA probe. In the control experiments, samples were treated with proteinase K (‘PK’) before electrophoresis, or the topoisomerase treatment was omitted, or both. The sizes of the (proteinase K-treated) linear pLUS892L DNA and the marker DNAs (‘M’) are indicated (in kb). (B) AFM examination of isolated DSS-cross-linked linear plasmid DNA (pLUS891L) without proteinase K-treatment (‘−PK’) shows supercoiled DNA structures that are held together by telomere–telomere interactions (left and center images). The center image shows a zoomed-in surface plot of the coiled DNA molecule boxed in the left image. Proteolytic digestion of the DSS-cross-linked DNA by proteinase K (‘+PK’) transformed it into relaxed, linear structures (right). The scale bar is 1 µm.

Figure 7.

Post-replicational Möbius strip-like structure. (A) The parental linear DNA molecule forms a circular configuration through associations between the TPs (black disks). The 3′-ends are indicated by the arrowheads. The association of the two 5′-ends is analogous to the half twist in a classical Möbius strip in the simplest form. (B) Replication is initiated at an internal origin and proceeds bidirectionally. The newly synthesized DNA strands are in gray. (C) Replication reaches the telomeres, and results in single-stranded gaps at the 3′-ends. ‘New’ TPs (gray disks) are posed to prime the patching synthesis. (D) The gaps are patched by DNA synthesis primed by new TPs. In this diagram, the ‘old’ TPs on the parental strands remain associated to each other, and the ‘new’ TPs on the daughter strands become associated with each other. This pseudo-dimeric structure resembles the single long strip that is produced, when a Möbius strip is cut along the centerline. It cannot be readily resolved into two monomers without rearranging non-covalent or covalent bonds through one of the two alternative pathways described below. (E) In the ‘TP exchange’ pathway, the parental TPs dissociate from each other and each pairs with the proper ‘daughter’ TP on the distal telomere of the same DNA. (F) In the ‘DNA exchange’ pathway, the structure is resolved by (presumably site-specific) recombination at the arbitrarily designated site (indicated by the arrows) without disruption of the TP associations. [Modified from reference (6)].