RecBCD, SbcCD and ExoI process a substrate created by convergent replisomes to complete DNA replication

Abstract

The accurate completion of DNA replication on the chromosome requires RecBCD and structure specific SbcCD and ExoI nucleases. However, the substrates and mechanism by which this reaction occurs remains unknown. Here we show that these completion enzymes operate on plasmid substrates containing two replisomes, but are not required for plasmids containing one replisome. Completion on the two-replisome plasmids requires RecBCD, but does not require RecA and no broken intermediates accumulate in its absence, indicating that the completion reaction occurs normally in the absence of any double-strand breaks. Further, similar to the chromosome, we show that when the normal completion reaction is prevented, an aberrant RecA-mediated recombination process leads to amplifications that drive most of the instabilities associated with the two-replisome substrates. The observations imply that the substrate SbcCD, ExoI and RecBCD act upon in vivo is created specifically by two convergent replisomes, and demonstrate that the function of RecBCD in completing replication is independent of double-strand break repair, and likely promotes joining of the strands of the convergent replication forks.

Figure 1.. Current model for completing DNA replication.

A) Convergent replication forks continue past their meeting point, creating a partially over-replicated substrate that contains three copies of the genetic information. B) SbcCD-ExoI recognize and cleave this branched over-replicated substrate which creates a DNA that can be resect and processed by RecBCD. Following resection, RecBCD promotes or recruit enzymes that join the convergent strands at the doubling point. C) When processing by SbcCD-ExoI is prevented or impaired, the over-replicated region persists, and an aberrant form of recombination then resolves the over-replicated region, leading to amplifications and genomic instabilities.

Figure 2.. Plasmids replicated by two-replisomes are less stable than those replicated by one replisome.

A) Diagram of the one-replisome and two replisome plasmids, containing a ColE1 and lambda origin or replication, respectively. B) In the absence of selection, the two-replisome plasmid is lost more rapidly than the one-replisome plasmid. Cultures containing the one-replisome (pBR322) or two-replisome (pCL01) plasmid were grown without selection. Serial dilutions of the culture were then plated with and without ampicillin selection to determine the fraction of cells that retained the plasmid over time. Error bars represent the standard error of 4 or more independent experiments. C) The instability of the two-replisome plasmid, relative to the one-replisome plasmid is not due a reduced growth rate of cells containing the two-replisome plasmid. The absorbance at 630 nm of cultures containing no plasmid, pBR322, or pCL01 grown at 37 °C is plotted over time. D) The instability of the two-replisome plasmid, relative to the one-replisome plasmid is not due to a reduced copy number during growth. A representative Southern analysis of the ampicillin resistance gene on the chromosome, on the plasmid pBR322, and on the plasmid pCL01 is shown. Total genomic DNA was purified from HL946 (Courcelle et al., 1999), containing a chromosomal copy of the ampicillin resistance gene, SR108 containing pBR322, and SR108 containing pCL01. Purified DNA was digested with EcoRV to linearize the plasmids and equal cell equivalents were then loaded and analyzed by Southern analysis using a P32-labeled ampicillin resistance gene as a probe. D) The copy number of each plasmid, relative to the chromosome is plotted. Error bars represent the standard error from five independent experiments. No plasmid (diamonds); pBR322 (squares); pCL01 (circles).

Figure 3.. Transformation of plasmids with two replisomes, but not one replisome, depends on the enzymes required to complete replication on the chromosome.

A) The transformation efficiency relative to wild type cells is shown for the strains indicated following electroporation of competent cells with 50ng of pBR322 (one-replisome). B) The transformation efficiency relative to wild type cells is shown for the strains indicated following electroporation of competent cells with 50ng of pCL01 (two-replisomes). Error bars represent the standard error of at least two independent experiments.

Figure 4.. Similar to completion on the chromosome, amplifications and instability on two-replisome plasmids are driven by an aberrant RecA-mediated form of recombination.

A) Inactivation of RecA restores the stability of the two-replisome plasmid to levels that approach that of the one-replisome plasmid. Cultures were treated as in Fig 2B. The fraction of cells retaining the one-replisome and two-replisome plasmid in wild type and recA mutants is plotted over time. Error bars represent the standard error of 4 independent experiments. WT pBR322 (filled squares); recA pBR322 (open squares); WT pCL01 (filled circles); recA pCL01 (open circles) B) Plasmid instability promoted by RecA correlates with an increased level of amplifications and aberrant multimeric species. Total genomic DNA from cells containing the one-replisome or two-replisome plasmid was purified and analyzed by southern analysis following agarose gel electrophoresis using P32-labeled pBR322 or pCL01 as a probe. (C) The fraction of unit-length monomeric plasmid is plotted for the one-replisome and two-replisome plasmid in the presence and absence of RecA. Error bars represent the standard error of four independent experiments.

Figure 5.. Stability of the two-replisome plasmid in various mutants.

A) The fraction of cells retaining the two-replisome plasmid for each strain is plotted over time. Cultures were treated as in Fig 2B. WT (filled circles); recA (open circles); recD (filled triangles); recG (filled inverted triangles); sbcCDxonA (diamonds); recBCsbcCDxonA (filled squares); Error bars represent the standard error of at least three independent experiments. B) A representative Southern analysis of plasmid pCL01 grown in each strain is shown. DNA was purified and analyzed as in Fig 4B.