Cellular characterization of the primosome and rep helicase in processing and restoration of replication following arrest by UV-induced DNA damage in Escherichia coli

Abstract

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.

Fig 1

Primosomal proteins and Rep do not act directly on UV damage-arrested replication forks. (A) [¹⁴C]thymine-labeled cultures were pulse labeled with [³H]thymidine for 5 s, filtered, rinsed, and resuspended in nonradioactive medium and immediately UV irradiated with 27 J/m². (B) The fraction of total DNA, ¹⁴C (○), and nascent DNA, ³H (■), remaining in the culture at each indicated time point is plotted. Graphs represent averages from at least three independent experiments. Error bars represent one standard deviation. The initial values for ³H and ¹⁴C ranged from 1,300 to 4,000 cpm and 700 to 2,100 cpm, respectively, for all experiments.

Fig 2

UV-arrested replication forks are processed normally in cells lacking priB, priC, or rep. (A) The predicted migration pattern of PvuII-digested pBR322 plasmid observed by two-dimensional agarose gel analysis and subsequent visualization with ³²P-labeled pBR322 is diagrammed. Nonreplicating plasmids run as a linear 4.4-kb fragment. Replicating plasmids form Y-shaped structures that migrate slower than nonreplicating DNA, forming an arc that extends above the linear region. Following UV irradiation, double-Y- or X-shaped intermediates are observed that migrate in the cone region behind the arc of Y-shaped molecules. (B) Two-dimensional agarose gels from wild-type, recF, priB302, priC, and rep cultures containing pBR322 at the indicated times following UV irradiation.

Fig 3

Primosomal proteins PriA and PriB, but not PriC, and Rep are required for the recovery of replication following 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 amount of total DNA at each time point relative to −10 min post-UV treatment, ¹⁴C (○), and DNA synthesis/2 min, ³H (□), is plotted. Graphs represent averages from at least three independent experiments. Error bars represent one standard deviation.

Fig 4

PriA and Rep are required for cell viability following UV irradiation. (A) The survival of wild-type (□), priA2 (▼), priB302 (△), priC (■), rep (♢), and recA (●) cultures after UV irradiation at the indicated doses. (B) The survival of wild-type (□), priA2 (▼), and recA (●) cultures replotted on a different scale. Graphs represent averages from at least three independent experiments. Error bars represent one standard deviation. A comparison of the viability of all strains across a range of UV doses used is shown in Fig. S2 in the supplemental material.

Fig 5

PriA is required for robust growth in the absence of DNA damage. (A) The OD₆₀₀ of wild-type (□), priA2 (▼), priB302 (△), priC (■), rep (♢), and recA (●) strains is plotted over time. (B) The number of viable cells in overnight cultures of wild-type, recA, priA2, priB302, priC, and rep strains is indicated in CFU × 10⁶/ml and CFU × 10⁶/OD.

Fig 6

Two models for primosome and Rep function following disruption by DNA damage. (A) A model proposing that PriA and Rep function specifically to reinitiate DNA synthesis following disruption events. (i) Following the disruption of the replication machinery (grayed circles) by DNA damage (∧), (ii) PriA or Rep functions in a reaction to transiently load DnaB and DnaG to prime the leading strand and then (iii) stably load DnaB and DnaG on the lagging strand (22, 27). (iv) The leading-strand primer allows for the de novo formation of an active replisome downstream from the site of disruption. (B) A model in which PriA and Rep are required by the replisome to maintain efficient replication. (i) Following disruption by DNA damage, the recovery of DNA synthesis requires that the lesion is either repaired (ii) or bypassed (iii) by translesion synthesis (not shown), as found in previous studies (13). (iv) Since PriA and Rep are needed to maintain replication in the absence of damage, PriA and Rep would also be required for an active replisome to be maintained once the replisome is reestablished and DNA synthesis resumes.