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...