R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1

Abstract

The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2). Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots. Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro. Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules. A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination. dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores. mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.

Fig. 1. 2-DE and EM analysis of intact plasmid mp1 in vivo replication and recombination intermediates. (A) Autoradiography of a 2-DE gel probed with mp1 DNA. Hybridization signals are explained in (B). Signals of unknown nature are marked with dashed lines and arrowheads. (C) Molecules from regions 1–8 were gently recovered from a gel. Enrichment of accumulated replication and recombination intermediates is shown. (D) One hundred molecules from each fraction were analysed by EM (for details see text). Bar = 0.5 kb. (E) Autoradiography of 2-DE gels of plasmid mp1 digested with the indicated restriction endonucleases. Hybridization patterns are explained schematically.

Fig. 2. Mapping of σ-like mp1 replication intermediates with one tail. Plasmid molecules from fractions 2, 3 and 4 were combined and linearized with BglI, PvuI and SmaI, respectively. EM investigation of 100 molecules each revealed symmetric simple Ys (A) and asymmetric simple Ys (B). The location of the origins dso1 and dso2 was deduced as described in the text. The x-axis corresponds to positions on the plasmid map as indicated in the model above. Examples are shown in the right panel. (C) Example and model of an uncut mp1 RC. Bar = 0.5 kb.

Fig. 3. Mapping of σ-like mp1 intermediates with two tails (double RCs). (A) Plasmid DNA from fractions 5 and 6 was combined and linearized with BglI, PvuI and SmaI, respectively. EM of 150 molecules in each digest revealed various double Ys. The location of the origins dso1 and dso2 was mapped. Examples are shown in the right panel. (B) Uncut double RCs initiated at dso1 and dso2 are explained in the model. Bar = 0.5 kb.

Fig. 4. Identification of in vivo R-loops in plasmid mp1. (A) EM mapping of 100 θ-like mp1 molecules. The x-axis corresponds to the plasmid map as indicated. (B) EM of uncut mp1 molecules with loops (arrowheads) that were incubated with SSB proteins. (C) Treatment of these molecules (arrowheads) with RNaseH resulted in the removal of the loops. Bar = 0.5 kb. (D) Physical and genetic map of mp1 depicting three putative ORFs. Arrowheads label the origins dso1 and dso2. (E) Northern blotting revealed three major transcripts with the sizes of ORF1–3 using mp1 DNA as probe. (F) Genetic organization of dso1 and ORF2 with a plant mt promotor consensus motif 5′ CGTA (Tracy and Stern, 1995; Binder et al., 1996). An alignment of dso1 to dsos of bacterial RC plasmids (Backert et al., 1998) and a Xer recombination core (Hodgman et al., 1998) is presented. Identical nucleotides are shaded in black and at least three identical residues are shaded in grey. Arrowheads indicate the RC nicking site.

Fig. 5. Analysis of early mp1 replication by an in vitro replication assay. (A) 2-DE of a construct harbouring the dso1 origin sequence, which was incubated in vitro for 60 min with a mitochondrial enzyme complex in the presence or absence of 10 µM NTPs. (B) Time course of mp1 in vitro replication using the dso1 construct and vector as a control. The results are the means of three experiments. (C) EM analysis of in vitro replication intermediates revealed R-loops (arrow) after 10 min. (D) Typical RCs were observed after 30 min. Bar = 0.5 kb. (E) Hypothetical model for the initiation of mp1 RCR involving an R-loop transcribed from ORF2 (see text).

Fig. 6. EM mapping of in vivo mp1 recombination intermediates. (A) DNA from fraction 8 was cut with BglI, PvuI and SmaI. EM of 100 molecules in each digest revealed X-shaped mp1 recombinants that mapped to dso1. Examples of cut (B) and uncut mp1 dimers (C) containing recombination junctions (arrowheads). Bar = 0.5 kb.

Fig. 7. EM of highly complex in vivo mp1 replication and recombination intermediates. (A) Molecules from fraction 8 were linearized with BglI, PvuI and SmaI, respectively. EM of 150 molecules mainly revealed symmetric simple Ys and double Ys. Examples are shown in the right panel. (B–F) Examples and explanation of uncut complex mp1 concatemers. (B) Open circle with a branched double-stranded tail of 1.3 kb. (C) Open circle with a 1.1 kb tail and a D-loop. (D) Highly branched mp1 molecule (total size 8.9 kb). (E) Model for the origin of this structure. The upper molecule may represent a double RC initiated at dso1 and dso2. Both RC tails are invaded by two other RCs (bottom). (F) Highly branched mp1 molecule (total size is 26.3 kb) that represents a double RC (reinitiated at dso1), recombining with a linear 10mer at dso1. Bars = 0.5 kb.

Fig. 8. Model for the resolution of mp1 concatemers. (A and B) Two representative examples of mp1 in vivo circles interconnected by a double-stranded tail. Hypothetical schemes depict a snap-back mechanism using invasion of the ends of the RC tails (grey circles) to release mp1 monomer units (1.3 kb). Bar = 0.5 kb. (C) Hypothetical model and enlarged sections of snap-back junctions (arrowheads) that were recently regarded as strand-switching intermediates (Backert, 2000). The arrow shows a free ssDNA end originating from the junction. Bar = 100 bases. (D) Mp1 concatemers produced by RCR are head-to-tail molecules with specific ends (dso1 or dso2). The 5′ overhang of the RC tail may invade the next dso to form a snap-back molecule. (E) After invasion, a replication fork (shaded grey) is formed in which the 3′ end extends. Resolution of this intermediate proceeds by nicking of the dso motif and strand exchange. This results in a Holliday structure that can easily be resolved by the recombination machinery (arrows). Finally, circular mp1 mono- or oligomers are released from the replicating RC that retains a protruding 3′ end (grey circle).

Fig. 9. Hypothetical model for the replication cycle of the mt plasmid mp1. Mp1 produces long dsDNA concatemers by a unique RC mechanism that is mainly initiated at origin dso1. During processing of the concatemers into single plasmid units, RCs or linear molecules with invasive 3′ ends are produced that allow invasion of other mp1 structures similar to phage T4. This model is compatible with structures mapped in this study.