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Endonuclease III (Endo III) and Formamidopyrimidine-N-glycosylase (Fpg) are two of the predominant DNA glycosylases in E. coli that remove oxidative base damage. In cell extracts and purified form, Endo III is generally more active on oxidized pyrimidines, while Fpg is more active towards oxidized purines. However, the substrate specificities of these enzymes partially overlap in vitro. Less is known about the relative contribution of these enzymes in restoring the genomic template following oxidative damage. In this study, we examined how efficiently Endo III and Fpg repair their oxidative substrates in vivo following treatment with hydrogen peroxide. We found that Fpg was nonredundant and required to rapidly remove its substrate lesions on the chromosome. In addition, Fpg also repaired a significant portion of the lesions recognized by Endo III, suggesting that it plays a prominent role in the global repair of both purine and pyrimidine damage in vivo. By comparison, Endo III did not affect the repair rate of Fpg substrates and was only responsible for repairing a subset of its own substrate lesions in vivo. The absence of Endo VIII or nucleotide excision repair did not significantly affect the global repair of either Fpg or Endo III substrates in vivo. Surprisingly, replication recovered after oxidative DNA damage in all mutants examined, even when lesions persisted in the DNA, suggesting the presence of an efficient mechanism to process or overcome oxidative damage encountered during replication.

Accurate replication in the presence of DNA damage is essential to genome stability and viability in all cells. In Escherichia coli, DNA replication forks blocked by UV-induced damage undergo a partial resection and RecF-catalyzed regression before synthesis resumes. These processing events generate distinct structural intermediates on the DNA that can be visualized in vivo using 2D agarose gels. However, the fate and behavior of the stalled replisome remains a central uncharacterized question. Here, we use thermosensitive mutants to show that the replisome's polymerases uncouple and transiently dissociate from the DNA in vivo. Inactivation of α, β, or τ subunits within the replisome is sufficient to signal and induce the RecF-mediated processing events observed following UV damage. By contrast, the helicase-primase complex (DnaB and DnaG) remains critically associated with the fork, leading to a loss of fork integrity, degradation, and aberrant intermediates when disrupted. The results reveal a dynamic replisome, capable of partial disassembly to allow access to the obstruction, while retaining subunits that maintain fork licensing and direct reassembly to the appropriate location after processing has occurred.replication fork processing | RecF pathway

Inaccurate replication in the presence of DNA damage is responsible for the majority of cellular rearrangements and mutagenesis observed in all cell types and is widely believed to be directly associated with the development of cancer in humans. DNA damage, such as that induced by UV irradiation, severely impairs the ability of replication to duplicate the genomic template accurately. A number of gene products have been identified that are required when replication encounters DNA lesions in the template. However, a remaining challenge has been to determine how these proteins process lesions during replication in vivo. Using Escherichia coli as a model system, we describe a procedure in which two-dimensional agarose-gel analysis can be used to identify the structural intermediates that arise on replicating plasmids in vivo following UV-induced DNA damage. This procedure has been used to demonstrate that replication forks blocked by UV-induced damage undergo a transient reversal that is stabilized by RecA and several gene products associated with the RecF pathway. The technique demonstrates that these replication intermediates are maintained until a time that correlates with the removal of the lesions by nucleotide excision repair and replication resumes. 4% glucose, 0.2% casamino acids, and 10 μg/ml thymine (DGCthy medium) and 100 μg/ml ampicillin is pelleted. The cell pellet is then resuspended in 200μl DGCthy medium lacking ampicillin and used to inoculate 20 ml of DGCthy medium. 2. Cultures are grown without ampicillin selection in a shaking incubator at 37°C to an OD600 of 0.5 (~5 x 10 8 cells/ml). Growth without ampicillin avoids selection against abnormal or unproductive replication intermediates that may arise in some mutants. In addition, if using UV light to induce damage, the removal of the ampicillin from the media is necessary because it absorbs strongly at these wavelengths and shields the cells, reducing the effective dose of UV. 3. Working under yellow lights, the culture is placed in a 15cm diameter Petri dish on a rotating platform for agitation. Our cultures are placed at a distance from a 15-watt germicidal lamp that produces an exposure rate of~1 J/m2/sec, which is measured using a UVC photometer. Cultures are irradiated with 50 J/m2 and then placed immediately back into the shaking 37°C incubator for the duration of the experiment. This dose produces, on average, 1 cyclobutane pyrimidine dimer every 4.5 kb of ssDNA . The yellow lighting prevents photoreactivation-reversal of cyclobutane pyrimidine dimers by photolyase.1. At times when replication intermediates are to be examined, a 0.75 ml aliquot of the culture is placed into 0.75 ml ice cold NET30 Buffer (100 mM NaCl, 10 mM Tris, pH 8.0, 30 mM EDTA) and placed on ice until the end of the time course. We typically run a 90 minute time course, with samples examined at 0, 15, 30, 45, 60, and 90 minutes. The EDTA and cold temperature serve to effectively stop replication and nucleotide excision repair. 2. Each sample is then pelleted, r...

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