Replication fork stalling and cell cycle arrest in UV-irradiated Escherichia coli

Abstract

Faithful duplication of the genome relies on the ability to cope with an imperfect template. We investigated replication of UV-damaged DNA in Escherichia coli and found that ongoing replication stops for at least 15-20 min before resuming. Undamaged origins of replication (oriC) continue to fire at the normal rate and in a DnaA-dependent manner. UV irradiation also induces substantial DnaA-independent replication. These two factors add substantially to the DNA synthesis detected after irradiation and together mask the delay in the progression of pre-existing forks in assays measuring net synthesis. All DNA synthesis after UV depends on DnaC, implying that replication restart of blocked forks requires DnaB loading and possibly the entire assembly of new replisomes. Restart appears to occur synchronously when most lesions have been removed. This raises the possibility that restart and lesion removal are coupled. Both restart and cell division suffer long delays if lesion removal is prevented, but restart can occur. Our data fit well with models invoking the stalling of replication forks and their extensive processing before replication can restart. Delayed restart avoids the dangers of excessive recombination that might result if forks skipped over lesion after lesion, leaving many gaps in their wake.

Figure 1.

DNA replication and replication restart after UV irradiation in E. coli. (A) Models of replication restart: (i) A fork skips over lesions (red triangles) leaving gaps in the nascent strands. (ii) A fork stalls at a leading strand block and reverses to form a Holliday-junction structure that is then processed to allow restart. (B) Diagram illustrating initiation and termination of chromosome replication. The open triangles indicate the positions of the lacO240 and tetO arrays used.

Figure 2.

Effect of UV on cell cycle progression. (A) Fluorescence microscopy showing replication of origin (red foci) and terminus (green foci) areas of the chromosome (combined phase contrast and fluorescence images are shown). The strain used was APS345. The incubation time after irradiation is indicated. (B) Enlargements of filaments from a repeat of the experiment in A. (C) Viable cell replication following irradiation. The strains used were MG1655 (wild type) and N5209 (sfiA11). Data for the irradiated cells are the mean (±SE) of three experiments. The data for the nonirradiated cells are the mean of two experiments that gave almost identical values.

Figure 3.

Effect of UV on DNA synthesis in dnaA46 and dnaC7 strains. (A) [³H]thymidine incorporation in wild-type (N1141) and dnaC7 (AU1080) cells. Data are the mean (±SE) of three experiments. (B) [³H]thymidine incorporation in wild-type (N1141) and dnaA46 (AU1068) cells. Data are the mean (±SE) of four to five experiments. The data for the wild type are reproduced from A for comparison. (C) [³H]thymidine incorporation in wild-type (N1141), dnaA167 (AU1093), and dnaA204 (AU1094) cells. Data are the mean (±SE) of three to four experiments. The data for the wild type are reproduced from A for comparison.

Figure 4.

Replication and repair of UV-irradiated DNA. (A) Schematic NotI restriction pattern of the E. coli chromosome. The distance from oriC to each end of the fragments is indicated. Fragments clockwise and counterclockwise of oriC are shown in red and blue, respectively. (B) Fluorograph showing BrdU incorporation into the chromosome of wild-type strain MG1655 ±UV. Origin-proximal bands labeled intensively are identified with green arrows. (C) BrdU incorporation pattern in dnaA46 strain AU1054. To inhibit oriC firing, cells were shifted to 42°C directly before adding BrdU. (D) Resolution of band I identified in B. (E) Pyrimidine dimer removal from strains MG1655 (wild type) and N4280 (uvrA).

Figure 5.

Effect of UV on DNA synthesis and chromosome replication. (A) [³H]thymidine incorporation in uvrA (AU1075) and uvrA dnaA46 (AU1072) cells. Data are the mean (±SE) of three experiments. Data for irradiated wild-type (N1141) cells (Fig. 3A) are included for comparison. (B) Changes in the origin to terminus ratio during incubation of irradiated and nonirradiated cells. The strain was RCe79 (dnaC7). Cells grown at 30°C were synchronized by incubation at 42°C for 45 min before irradiation and shifting back to 30°C. Data are the mean (±SE) of three experiments.

Figure 6.

Effect of UV lesions on cell cycle progression and DNA synthesis in the absence of DNA excision repair. (A) Fluorescence microscopy showing replication of origin (red foci) and terminus (green foci) areas of the chromosome. The strain was RCe129 (uvrA). (B) Changes in the origin to terminus ratio during incubation of irradiated and nonirradiated cells. The strain was RCe120 (dnaC7 uvrA). Cells grown at 30°C were synchronized by incubation at 42°C for 45 min before irradiation and shifting back to 30°C. Data are the mean (±SE) of three or more experiments. (C) Fluorograph showing the time course and pattern of BrdU incorporation into the chromosome of uvrA strain N4280 with or without UV irradiation as indicated.