Replication slippage involves DNA polymerase pausing and dissociation

Abstract

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

Fig. 1. Experimental system. Schematic structure of the plasmid and of the primer extension reaction used in this work. The recombination unit of plasmids FX, FXb and FXc consists of two 27 bp DRs (black arrows), flanking a pair of 300 bp IRs (grey arrows) and a central 1370 bp region (insert).

Fig. 2. Comparison of slippage efficiency of three different DNA structures. (A) Schematic representation of the base of the hairpin of each structure. DRs are represented by black arrows and the GC clamp by a hatched box. (B) Analysis of primer extension products by agarose gel electrophoresis. Assays were carried out as described in Materials and methods in the presence of 25 ng of labelled primed template, 1 U of T7 pol and decreasing amounts of SSB. Templates used were FX, FXb and FXc, as indicated at the top of the panel. Lanes 1, 750 ng of SSB (10 times more than the saturating amount); lanes 2, 375 ng of SSB (five times the saturating amount); lanes 3, 75 ng of SSB (saturating amount); lanes 4, 7.5 ng of SSB (10 times less than the saturating amount); lanes 5, no SSB. P, H and S refer to parental, heteroduplex and stalled molecules, respectively. (C) Analysis of primer extension products by denaturing polyacrylamide gel electro phoresis. Precise characterization of the products shown in (B) was done by restriction analysis and determination of the size of the different DNA fragments. Samples are in the same order as in (B). The products were cleaved with XmnI and RsaI. To the right of the figure are schematized the structures of the expected products with the relevant restriction sites (X for XmnI and R for RsaI). The numbers in bold indicate the sizes of the informative restriction segments; the size of the P segment (518) is that for FX; the two other templates, FXb and FXc, yield a segment of 508 bases. To the left of the figure are indicated the positions of migration of these segments. The ladder on the right of the gel corresponds to the sequence of M13mp18 with primer –40.

Fig. 3. Stalled molecules can be converted into either heteroduplex or parental molecules. Primer extension was carried out as described in Materials and methods in the presence of 7.5 U of T7 pol, 375 ng of labelled primed template FXc and different amounts of SSB. Aliquots were withdrawn at the times indicated at the top of each panel and loaded on agarose gels. (A) A 1.12 µg aliquot of SSB (saturating amount). (B) An 11.2 µg aliquot of SSB (10 times more than the saturating amount). (C) A 112.5 ng aliquot of SSB at the beginning of the reaction followed by the addition of 2.6 µg after 4 min. (D) Schematic representation of the base of the hairpin showing the possible evolution of stalled molecules. DRs are represented by black arrows and the GC clamp by a hatched box. As above, P, H and S refer to parental, heteroduplex and stalled molecules, respectively, whereas A stands for abortive products (see text for details).

Fig. 4. Analysis of stalled molecules on two different DNA structures. Primer extension was carried out as described in Materials and methods in the presence of 375 ng of labelled primed template FXb (A and C) or FXc (B and D), 1.12 µg of SSB (saturating amount) and 7.5 U of T7 pol. Aliquots were withdrawn at the times indicated at the top of each panel and loaded on either agarose gels (A and B) or sequencing gels (C and D). An interpretation of the results is schematized in (E) and (F) for each structure (only the bases of the hairpins are represented). DRs are represented by black arrows, the GC clamp by a hatched box and the annealing event by a dotted arrow. In (E), only one replication intermediate is represented because only one main stop position (at the end of the DR) was found. In (F), several replication intermediates are represented because multiple stops occurred inside the DR. See text for details.

Fig. 5. Stalled molecules accumulate in the presence of a DNA polymerase trapping agent. (A) Primer extension was carried out as described in Materials and methods in the presence of 5 U of T7 pol, 250 ng of labelled primed template FXc, 75 ng of SSB (10 times less than the saturating amount) and three deoxynucleotides (dATP, dCTP and dGTP). The reaction was started by the simultaneous addition of the fourth deoxynucleotide (dTTP) and 5–10 µg of heparin. Aliquots were withdrawn at the times indicated at the top of each well and loaded on an agarose gel. After 4 min, 2.25 µg of SSB were added (five times more than the saturating amount), and 10 µl aliquots were withdrawn until 25 min. The last lane is a control sample incubated for 25 min in the presence of 0.5 U of T7 pol, 25 ng of primed template FXc, 375 ng of SSB and the four dNTPs, but in the absence of heparin. p/t refers to the primer–template. (B) Schematic representation of the base of the hairpin showing the interpretation of the results. In the presence of heparin, the polymerase replicates 1.2 kb until encounter ing the hairpin, where it dissociates. DRs are represented by black arrows, the GC clamp by a hatched box, the labelled primer by an asterisk and the DNA polymerase by a sphere. The cross on the sphere indicates the trapping of the dissociated polymerase by heparin.

Fig. 6. Effect of the length of the DR on replication slippage. (A) A primer extension assay was carried out as described in Materials and methods in the presence of 25 ng of labelled primed template, 75 ng of SSB (saturating amount) and either 0.5 U of T7 pol (lanes 1–3) or 10 U of pol III HE (lanes 4–6). DNA templates were FXb-14 (lanes 1 and 4), FXb-27 (lanes 2 and 5) and FXb-55 (lanes 3 and 6), having DRs of 14, 27 and 55 bp, respectively. (B) Schematic representation of the base of the hairpin indicating the interpretation of the results. Left, short DR (black arrows); right, longer DR (longer black arrows). Dotted arrows represent why increasing the length of the DR increases the distance for the tip of the newly synthesized strand to pair to the second DR after unpairing from the first DR.

Fig. 7. Schematic representation of the slippage process at a replication fork. During DNA synthesis of a repeated sequence (step 1), the polymerase reaches a barrier on the lagging strand (a hairpin structure in our experimental system) and pauses (step 2). Polymerase dissociation occurs (step 3). If the polymerase is not able to disrupt the barrier (with its strand displacement activity), then the tip of the newly synthesized strand can unpair from its template and anneal to the second repeat beyond the barrier, allowing polymerase re-loading and resumption of the synthesis (step 4).