Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo

Abstract

Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase-DNA cleavage complexes, which are apparently transformed into double-stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two-dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli. Restriction endonuclease-digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone-induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double-stranded breaks by an indirect mechanism after quinolone treatment.

Fig. 1

Norfloxacin treatment causes cleavage complex formation at gyrase binding sites. Cells containing pBR322 (lanes 1–10) or pBR322MUT990 (lane 11) were lysed under cleavage (C) or resealing (R) conditions; DNA was extracted and dialysed overnight into TE buffer and then digested with EcoRI. Samples were loaded onto a 1.2% agarose gel, which was analysed by Southern hybridization using a pBR322 probe. A. JH39 (lanes 1–3), parC^R (lanes 4–6), or gyrA^R (lanes 7–9) cells were treated with norfloxacin (NOR) for 6 min (lanes 2–3, lanes 5–6, lanes 8–9) or were untreated (lanes 1, 4 and 7). The marker lane consisted of a mixture of restriction fragments derived from plasmids pBR322 and pSF5 (a pBR322 derivative with an approximately 13-kb fragment of phage lambda DNA; obtained from Dr M. Feiss, University of Iowa). B. JH39 cells carrying pBR322 (lane 10) or pBR322MUT990 (lane 11) were treated with norfloxacin and DNA was extracted under cleavage lysis conditions. Arrows indicate cleavage complex bands resulting from cleavage at the major gyrase binding site centred at 990 bp. The marker lane consisted of a mixture of restriction fragments of pBR322.

Fig. 2

Diagrams of pBR322 DNA after cleavage with AlwNI (A) or EcoRI (B). Replication of pBR322 begins unidirectionally at position 2535 and is indicated by the thick black arrow. AlwNI cuts just behind the origin at position 2890 and EcoRI cuts midway around the plasmid at position 1, as indicated in the schematic on top. The major gyrase binding site is at position 990 and is indicated by two overlapping ovals. The map indicates the forms of DNA produced as replication proceeds from 1.0× to 2.0×. Non-replicating linear (LM) monomers are 4361 base pairs. As the replication fork crosses the location of the restriction endonuclease recognition site, the resulting molecules change from bubble-form (B) to double-Y form (DY), by passing through a simple-Y form [expected as a spot along the simple-Y arc (Y)]. The bubble-to-double-Y transition occurs at 1.9× for AlwNI and 1.6× for EcoRI. At 1.35×, the replication fork reaches the gyrase binding site centred at 990 bp, where stalling can occur if a gyrase cleavage complex is bound (molecules indicated by an asterisk). To the right of the maps are illustrations of the expected 2D gel electrophoresis patterns. The location of the entire simple-Y arc is shown as a dotted line; this arc is not expected for normal pBR322 replication (except for the discrete spot at the bubble-to-double-Y transition).

Fig. 3

Norfloxacin treatment causes stalled replication forks at gyrase binding sites. DNA was isolated from JH39 cells carrying pBR322 (upper panels) or pBR322MUT990 (lower panels) and digested with AlwNI. The samples were as follows: A. Cells were untreated (UTR) and DNA was isolated under cleavage conditions (CLV). B. Cells were treated with norfloxacin (NOR) and DNA was isolated under cleavage conditions. C. Cells were treated with norfloxacin and DNA was isolated under resealing conditions (RSL). D. Shorter exposures of panel C. E. Cells were untreated and DNA was isolated under resealing conditions (upper panel). The illustration (E, lower) indicates the various arcs seen after AlwNI digestion where B, bubble; LM, linear monomer; Y, simple-Y; AY, asymmetric-Y; DY, double-Y; X, X-shaped molecules; OC, open circular monomer. The arrows indicate the location that corresponds to the asterisk in the lower panel of (E).

Fig. 4

DNA molecules resulting from in vitro resealing or cleavage lysis conditions after norfloxacin treatment (A) and DNA molecules resulting from breakage of sigma molecules in vivo (B). The partially replicated plasmid considered in this figure is stalled at the major gyrase binding site (centred at 990 bp). The gyrase cleavage complex is indicated by red overlapping ovals. Newly synthesized DNA strands are shown in green. The purple and green arrowheads indicate the AlwNI and EcoRI cut sites respectively. A. Reactions that occur during in vitro lysis are as follows. Under resealing lysis conditions, a gyrase cleavage complex at a blocked fork would reseal the latent DNA break, resulting in an intact covalently closed circular replication intermediate (CCC RI) (arrow 1); treatment of this molecule with either AlwNI or EcoRI yields a bubble. Under cleavage conditions, the latent DNA break within the gyrase cleavage complex at a stalled fork is revealed, resulting in a simple-Y (Y) molecule (arrow 2); treatment of this molecule with either AlwNI or EcoRI yields a linear fragment plus a shortened simple-Y (SSY) molecule. If a gyrase cleavage complex exists well ahead of the replication fork, isolation under resealing conditions results in an intact CCC RI (arrow 3); treatment of this molecule with either AlwNI or EcoRI yields a bubble. In this case, isolation under cleavage conditions results in a bubble (B) (arrow 4); treatment of this molecule with either AlwNI or EcoRI yields a linear fragment plus a shortened bubble. If a gyrase cleavage complex is located behind the replication fork, isolation under resealing conditions results in an intact CCC RI (arrow 5); treatment of this molecule with either AlwNI or EcoRI yields a bubble. In this case, isolation under cleavage conditions produces a two-tailed circle (TTC) (arrow 6); treatment of this molecule with either AlwNI or EcoRI yields a double-Y (DY) molecule. B. A CCC RI without a gyrase cleavage complex can be broken in vivo at the fixed branch (left side) or the replicating fork (right side).

Fig. 5

Norfloxacin treatment causes formation of shortened simple-Y molecules. DNA was isolated from JH39 cells carrying pBR322 and digested with EcoRI. The samples were as follows: A. DNA was isolated under cleavage conditions (CLV) from untreated cells (UTR). B. DNA was isolated under cleavage conditions from norfloxacin-treated cells (NOR). C. DNA was isolated under resealing conditions (RSL) from norfloxacin-treated cells. The illustrations below indicate the various arcs seen after EcoRI digestion, where B, bubble; LM, linear monomer; SSY, shortened simple-Y; Y, simple-Y; DY, double-Y; AY, asymmetric-Y; and OC, open circular monomer.

Fig. 6

Norfloxacin treatment causes formation of a sigma arc in undigested DNA. pBR322 DNA was isolated from JH39 under the following conditions: A. Cleavage conditions (CLV) from untreated cells (UTR). B. Cleavage conditions from norfloxacin-treated cells (NOR). C. Resealing conditions (RSL) from norfloxacin-treated cells. None of the samples were treated with restriction enzymes. The illustrations indicate the various arcs, where B, bubble (blue); LM, linear monomer; LD, linear dimer; Y, simple-Y (green); TTC, two-tailed circle; S, sigma (red); OC, open circular monomer; CCC, covalently closed circle; CCC RI, covalently closed circle replication intermediate; TopCCC, topoisomers of CCCs; CCC2, dimer of CCC; and CCC/OC, CCC in first dimension, OC in second dimension.

Fig. 7

Rolling circle replication in recB cells in the absence of norfloxacin. pBR322 DNA was isolated under cleavage conditions (CLV) from recB⁻ cells without drug treatment (UTR). DNA was undigested (A) or digested with EcoRI (B).

Fig. 8

Model for fork blockage and breakage in vivo. Replication is initiated at the unidirectional origin and the fork encounters a norfloxacin-stabilized cleavage complex, resulting in fork blockage. Once the fork is blocked, the cleavage complex can reverse in vivo, presumably allowing for direct fork restart. Alternatively, the blocked replication fork can be processed by in vivo fork breakage (at either the fixed branch or the active fork).