Abasic sites and strand breaks in DNA cause transcriptional mutagenesis in Escherichia coli

Abstract

DNA damage occurs continuously, and faithful replication and transcription are essential for maintaining cell viability. Cells in nature are not dividing and replicating DNA often; therefore it is important to consider the outcome of RNA polymerase (RNAP) encounters with DNA damage. Base damage in the DNA can affect transcriptional fidelity, leading to production of mutant mRNA and protein in a process termed transcriptional mutagenesis (TM). Abasic (AP) sites and strand breaks are frequently occurring, spontaneous damages that are also base excision repair (BER) intermediates. In vitro studies have demonstrated that these lesions can be bypassed by RNAP; however this has never been assessed in vivo. This study demonstrates that RNAP is capable of bypassing AP sites and strand breaks in Escherichia coli and results in TM through adenine incorporation in nascent mRNA. Elimination of the enzymes that process these lesions further increases TM; however, such mutants can still complete repair by other downstream pathways. These results show that AP sites and strand breaks can result in mutagenic RNAP bypass and have important implications for the biologic endpoints of DNA damage.ost cells in nature are in a state of limited growth and are therefore not often engaged in replication (1, 2). Thus, the functional viability of most types of cells likely depends more on faithful transcription and translation than on faithful replication. Several base damages are bypassed in vivo by RNA polymerase (RNAP) in a mutagenic manner, resulting in transcriptional mutagenesis (TM) (3-7). In nondividing cell populations, TM could have a large contribution to the mutant protein burden and resulting cellular phenotype, compared with replicationbased mutagenesis events.Base excision repair (BER) is responsible for the processing of many small lesions, and in particular, those that do not cause significant helix distortion. During BER these lesions are removed by specialized DNA N-glycosylases. Monofunctional glycosylases remove the damaged base, leaving an abasic (AP) site. Bifunctional glycosylase/AP lyase enzymes remove the lesion and nick the DNA strand via β-or β,δ-elimination, resulting in a 3′ blocking group (8). Next, AP endonucleases process the AP sites and 3′ blocking groups to produce 3′-termini compatible with subsequent DNA repair synthesis (8, 9). In vitro, RNAP can bypass several types of repair intermediates, including AP sites and various strand breaks (10-14). Whether RNAP is capable of bypassing abasic sites and strand breaks in live organisms is an important issue for defining the spectrum of DNA damages that can cause TM, as well as for understanding the relationships between RNAP and DNA repair processes. Here we have demonstrated that RNAP is capable of bypassing AP sites and strand breaks in Escherichia coli. ResultsWe investigated TM mediated by AP sites and strand breaks by using damage-tailored plasmid constructs and using a transcriptional mutagenesis luciferase assay system (TM-LAS) (3, 15) (Fig. S1) in iso...

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“…However, in nonreplicating cells, AP sites are also the source of transcriptional mutagenesis (TM). In this respect, Clauson et al (49) reported that nonreplicating cells of a ⌬nfo exoA E. coli strain increased the levels of TM in response to the presence of AP sites, single-strand breaks, and oxidized bases such as 8-oxo-G. In B. subtilis, a variant of this process has been shown to be involved in adaptive mutagenesis and has been termed transcription-associated mutagenesis (50,51).…”

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“…A s one of the most frequent lesions in the genome, DNA abasic (AP) sites are derived from either spontaneous hydrolysis of the glycosidic bonds, often as a result of alkylation or oxidation of purines, or the enzymatic removal of modified bases by glycosylase enzymes in the base excision repair (BER) pathway (1)(2)(3). Failure to repair AP sites poses a severe challenge to polymerases and also leads to strand breaks, DNA cross-links, and transcriptional mutations, resulting in cellular dysfunction (1,2,4).…”

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“…1 These authors contributed equally to this work. specific post-translational modifications, oligomeric state and/or subcellular localization, which determine the binding partners (Copley, 2012;Sirover, 2005Sirover, , 2011. Each function requires binding into specific protein complexes.…”