Nucleotide excision repair or polymerase V-mediated lesion bypass can act to restore UV-arrested replication forks in Escherichia coli

Abstract

Nucleotide excision repair and translesion DNA synthesis are two processes that operate at arrested replication forks to reduce the frequency of recombination and promote cell survival following UV-induced DNA damage. While nucleotide excision repair is generally considered to be error free, translesion synthesis can result in mutations, making it important to identify the order and conditions that determine when each process is recruited to the arrested fork. We show here that at early times following UV irradiation, the recovery of DNA synthesis occurs through nucleotide excision repair of the lesion. In the absence of repair or when the repair capacity of the cell has been exceeded, translesion synthesis by polymerase V (Pol V) allows DNA synthesis to resume and is required to protect the arrested replication fork from degradation. Pol II and Pol IV do not contribute detectably to survival, mutagenesis, or restoration of DNA synthesis, suggesting that, in vivo, these polymerases are not functionally redundant with Pol V at UV-induced lesions. We discuss a model in which cells first use DNA repair to process replication-arresting UV lesions before resorting to mutagenic pathways such as translesion DNA synthesis to bypass these impediments to replication progression.

FIG. 1.

Pol V is required for resistance and mutagenesis following UV irradiation. (A) The survival of parental (▪), polB (▴), dinB (□), umuDC (•), umuC (⋄), uvrA (○), polB dinB umuDC (▵), and polB dinB umuDC uvrA (♦) cultures is shown after UV irradiation at the indicated doses. (B) The survival of parental (▪), uvrA (○), and polB dinB umuDC uvrA (♦) cultures replotted on a different scale. Graphs represent an average of at least three independent experiments. Error bars represent 1 standard deviation. (C) Cultures were irradiated at the indicated doses and examined for the number of rifampin (Rif)-resistant colonies that appeared following an overnight incubation. The number of rifampin-resistant colonies that appeared per 10⁸ cells is plotted. Graphs represent an average of four independent experiments. Error bars represent 1 standard deviation.

FIG. 2.

Nucleotide excision repair, but not translesion DNA synthesis, is required for the recovery of DNA replication after UV irradiation. [³H]thymidine was added to [¹⁴C]thymine-prelabeled cultures for 2 min at the indicated times following either 27 J/m² UV irradiation (filled symbols) or mock irradiation (open symbols) at time zero. The relative amounts of total DNA (¹⁴C; ○) and DNA synthesis/2 min (³H; □) are plotted. Graphs represent an average of at least three independent experiments. Error bars represent 1 standard deviation.

FIG. 3.

Increased degradation occurs at the growing fork after irradiation in polB dinB umuDC uvrA cells. [³H]thymidine was added to [¹⁴C]thymine-prelabeled cells for 5 s prior to irradiation with 27 J/m² in nonlabeled medium. The fraction of radioactive nucleotides remaining in the DNA is plotted over time. The initial values for ³H and ¹⁴C were between 2,500 to 4,000 and 1,200 to 1,700 cpm, respectively, for all experiments. Graphs represent an average of at least three independent experiments. Error bars represent 1 standard deviation. Results for total DNA (¹⁴C; □) and nascent DNA (³H; ▪) are shown.

FIG. 4.

Nucleotide excision repair and translesion DNA synthesis are required for nascent DNA gap filling. The size of the DNA synthesized immediately after irradiation was analyzed by alkali sucrose gradients over time. [¹⁴C]thymine-prelabeled cells were irradiated at a dose of 27 J/m², pulse-labeled with [³H]thymidine for 5 min, and then filtered into nonlabeled medium. Cultures collected immediately after ³H labeling are referred to here as time zero. Amounts of ³H and ¹⁴C in each fraction are plotted as a percentage of the total counts in each gradient. ³H and ¹⁴C values were between 4,200 to 9,500 and 1,500 to 4,300 cpm per gradient, respectively. Results for DNA synthesized before irradiation (¹⁴C; □) and DNA synthesized after treatment (³H; •) are shown. Graphs represent one of at least three independent experiments.

FIG. 5.

Pol V contributes to the rate that DNA synthesis resumes, protection of the replication fork in the absence of repair, and daughter-strand gap repair after UV irradiation. (A) Data were obtained and plotted as in Fig. 2. Each graph represents an average of at least three independent experiments. Error bars represent 1 standard deviation. Total DNA (¹⁴C) in mock-irradiated cultures (○), total DNA in irradiated cultures (•), the rate of DNA synthesis (³H) in mock-irradiated cultures (□), and the rate of DNA synthesis in irradiated cultures (▪) are shown. (B) Data were obtained and plotted as in Fig. 3. The initial values for ³H and ¹⁴C were between 750 to 1,200 and 800 to 1,200 cpm, respectively, for all experiments. Results for total DNA (¹⁴C; □) and nascent DNA (³H; ▪) are shown. Each graph represents an average of at least three independent experiments. Error bars represent 1 standard deviation. (C) Data were obtained and plotted as in Fig. 4. ³H and ¹⁴C values were between 6,700 to 11,000 and 2,500 to 4,000 cpm per gradient, respectively. Results for DNA synthesized before irradiation (¹⁴C; ▪) and DNA synthesized after treatment (³H; •) are shown. Graphs represent one of at least three independent experiments.