DNA replication triggered by double-stranded breaks in E. coli: dependence on homologous recombination functions

Abstract

Homologous recombination-dependent DNA replication (RDR) of a lambda cos site-carrying plasmid is demonstrated in E. coli cells when the cells express lambda terminase that introduces a double-stranded break into the cos site. RDR occurs in normal wild-type cells if the plasmid also contains the recombination hotspot chi. Chi is dispensable when cells are induced for the SOS response or contain a recD mutation. recBC sbcA mutant cells are also capable of RDR induction. A recN mutation greatly reduces RDR in normal cells, but not in SOS-induced cells. RDR proceeds by the theta mode or rolling circle mode of DNA synthesis, yielding covalently closed circular plasmid monomers or linear plasmid multimers, respectively. Previously described inducible stable DNA replication is considered to be a special type of RDR that starts exclusively from specific sites (oriMs) on the chromosome.

Figure 1. Maps of Plasmids Containing λ Terminase Genes or a cos Site

The position and direction of promoters on pFM123 relevant to the regulated expression of the terminase genes Nu1 and A are shown by arrows. P_R and P_L are the λ early rightward and leftward promoters, respectively, and P_RM is the promoter for the gene cl necessary for the maintenance of a repressed prophage. W, B, and C are the genes located at the left end region of λ. bla is the β-lactamase gene. See Murialdo (1988) for details of the construction of pFM123. The unique EcoRI site in pHK-cos is shown on the map. DNA fragments containing the χ and Ω sequences were inserted into this site to generate pHKχ1-cos and pHKχ3-cos. The closed region in pHK-cos, pHKχ1-cos, and pHKχ3-cos represents the λ fragment (∼450 bp) carrying a cos site (see Experimental Procedures). The terminase-cutting site is located around the middle of the fragment, and in these plasmids the lacZ′ gene is proximal to the right end sequence of λ. Therefore, RecBCD enzyme recognizing a DSB generated at these cos sites travels counterclockwise (see text). An arrow in pHKχ1-cos and pHKχ3-cos represents the χ sequence, and the arrowhead indicates the 3′ end of the sequence. Abbreviations and symbols: kan, the kanamycin resistance gene; ori, the plasmid replication origin; prime and capital delta, the truncation and internal deletion of genes, respectively. The figures are not drawn to scale.

Figure 2. The Expected Reactions of DSB-Triggered DNA Replication

The closed region in circular plasmids represents a λ fragment carrying a cos site. λ terminase molecules are shown by hatched squares. Arrowheads represent the 3′ ends of DNA strands. Supercoiling of circular molecules is ignored. See text for details.

Figure 3. The Copy Number Increase of pHK-cos and pHK in the Presence of Rif and Cm in rec⁺ sbc⁺ Cells

(A) An autoradiogram of Southern blot hybridization to determine replication of pHK-cos after SOS induction. AQ5004 (rec⁺ sbc⁺ thyA) carrying pHK-cos and pFM123 was grown to 2.5 × 10⁸ cells/ml in M9-CAA medium containing [³H]thymine (2 µCi/ml) and treated as described in the text. A mixture of thymine (8 µg/ml), Rif (200 µg/ml), and Cm (150 µg/ml) was added at time 0. Origin, the origin of electrophoresis migration; oc, open circular plasmid molecules. Duplicated samples were withdrawn at time 0 and processed independently. (B) The Southern blot hybridization filter shown in (A) was exposed to a screen for a phosphorimager, and the intensity of ³²P radioactivity in each ccc DNA band was determined. The relative increase obtained from the result is shown by closed circles. AQ5004 carrying pHK-cos and pBR322 or pHK and pFM123 was also grown as described above and was induced for the SOS response. The relative increase of ccc molecules of pHK-cos and pHK in the presence of Rif and Cm is shown by closed and open triangles, respectively. Open circles are the relative increase of pHK-cos in uninduced AQ5004 cells in the presence of pFM123.

Figure 4. The Copy Number Increase of pHKχ1-cos, pHKχ3-cos, and pHK-cos in the Presence of pFM123 in Uninduced Wild-Type and rec Mutant Cells

Cells were grown and treated as described in the text and Figure 3. (A) An autoradiogram of Southern blot hybridization to determine replication of pHKχ1-cos in AQ5004 (rec⁺ sbc⁺). (B) The relative copy number increase of pHKχ1-cos (closed circles), pHKχ3-cos (open triangles), and pHK-cos (open circles) in AQ5004 and of pHK-cos (closed triangles) in AQ9601 (recD). The result of pHKχ1-cos was obtained from the experiment shown in (A). (C) The relative copy number increase of pHKχ1-cos in AQ9450 (recA, open circles) and AQ9169 (recBC, closed circles).

Figure 5. The Copy Number Increase of the ccc Molecules and LPMs of pHK-cos in Cells Carrying recB and recC Mutations

(A and B) Autoradiograms of Southern blot hybridization to determine replication of pHK-cos in recBC sbcA and recBC sbcA recE mutant cells. AQ3625 (A) and AQ3626 (B) carrying pHK-cos and pFM123 were grown to 2.5 × 10⁸ cells/ml in the presence of [³H]thymidine (2 µCi/ml), thymidine (10 µg/ml), and 2′-deoxyadenosine (300 µg/ml) and treated as described in the text. Rif and Cm were added at time 0. (C) The relative increase of ccc molecules (closed circles) and LPMs (bold x’s) of pHK-cos in AQ3625 (A). AQ3625 carrying pHK-cos and pBR322 was also grown as described above, and the relative increase of ccc molecules of pHK-cos (open circles) was determined by the same method. (D) The relative increase of ccc molecules of pHK-cos in the presence of pFM123 was determined as described above in the following strains: AQ9169 (recBC, open triangles), AQ3626 (recBC sbcA recE, closed triangles), AQ9561 (recBC sbcA recA, open circles). The result of AQ3626 was obtained from the experiment shown in (B).

Figure 6. The Relative Copy Number Increase of ccc Molecules of pHKχ1-cos in Wild-Type and recN Mutant Cells

AQ5004 (recN⁺ thyA) and AQ9717 (recN thyA) carrying pHKχ1-cos and pFM123 were grown and treated as described in the text and Figure 3. The results are the relative increase of ccc molecules of pHKχ1-cos in uninduced (A) and SOS-induced (B) cells. Open symbols, AQ5004; closed symbols, AQ9717.

Figure 7. A Model for the Initiation Mechanism of RDR

A part of an intact circular DNA molecule (thin line), an invading ssDNA tail (thick line), a primer RNA molecule (wavy line), and a newly synthesized DNA fragment (broken line) are shown. A stem–loop structure on the displaced ssDNA in (A) represents a signal for DnaB loading. A DnaB hexamer and its direction of translocation on ssDNA are shown by a thick arrow. DnaG primase is represented by a stippled circle. Small arrowheads indicate the 3′ ends of DNA strands. See text for details.

Figure 8. A Model for DSB Repair in Wild-Type Cells

The E. coli chromosome and a newly synthesized DNA fragment are shown by thin double lines and a broken line, respectively. A χ site is denoted by a small arrow. Arrowheads represent the 3′ ends of DNA strands and of χ sequences. DnaB helicase and DnaG primer are shown by the same symbols as in Figure 7. See text for details of the model.

Figure 9. Restoration of a Destroyed Replication Fork by RDR

The E. coli chromosome and a newly synthesized DNA fragment are as in Figure 8. A small arrow represents a χ site. Arrowheads indicate the 3′ ends of DNA strands and of the χ sequence. DnaB helicase and DnaG primer are shown by the same symbols as in Figure 7. See text for details.