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. 2010 Feb 23;107(8):3657-62.
doi: 10.1073/pnas.0913191107. Epub 2010 Feb 8.

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

Affiliations

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

Cheryl L Clauson et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
AP site-mediated transcriptional mutagenesis in vivo. (A) AB1157 xth nfo cells were transformed with URA/ST (open circles), APTHF/ST (closed triangles), AP/ST (open triangles), or ST/ST (closed circles) construct, allowed a 30-min recovery from electroporation, and TM level (expressed as relative light units or RLUs) was assayed by TM-LAS (Fig. S1) at 0, 45, 120, and 240 min following IPTG initiation of transcription. Each point represents the mean of at least three replicates ± SEM. (B) AB1157 xth nfo nfi cells were transformed with designated damage-containing constructs, followed by a 30-min recovery from electroporation; then, at indicated times following luciferase induction, RNA was extracted to determine AP site-driven TM events. *In the AP/ST 5-min experiment, cells were immediately resuspended in medium containing IPTG following electroporation, and RNA was isolated 5 min later. RT-PCR and subsequent cDNA sequencing allowed determination of the ribonucleotide incorporated during RNAP bypass. The number of insertion events detected for each ribonucleotide is indicated for either adenine or uracil, with the percentage of the total in parentheses. Each number represents combined sequencing results from two independent transformation events with two independent preparations of construct. Uracil at the first position of codon 445 results from transcription of repaired molecules and yields a Stop codon (producing inactive luciferase), except in the case of APTHF/WT, in which repaired constructs would direct adenine insertion at the first position of codon 445. Adenine insertion results from RNAP bypass over the AP site and yields a Lys codon (producing active luciferase).
Fig. 2.
Fig. 2.
Strand break–mediated transcriptional mutagenesis in vivo. (A) AB1157 xth nfo cells were transformed with 8OG/ST (open circles), SSB/ST (closed triangles) or ST/ST (closed circles) construct, allowed a 30-min recovery from electroporation, and TM level (expressed in relative light units or RLUs) for these transformed cells was measured as before (Fig. 1). (B) AB1157 xth nfo nfi cells were transformed with indicated damage-containing constructs, and RNA extracted to determine strand break-driven TM as before (Fig. 1).
Fig. 3.
Fig. 3.
RNAP bypass of AP sites and strand breaks reveal the relationship between the transcriptional machinery and DNA repair processes. Nonbulky lesions (small green box) in DNA cause transcriptional mutagenesis (TM) and are primarily repaired by enzymes in the base excision repair (BER) pathway. DNA N-glycosylases remove base lesions from the DNA, generating an abasic site (monofunctional glycosylase) or a strand break (bifunctional glycosylase/AP-lyase). Such BER intermediates also cause TM, where RNAP bypass results in transcripts containing adenine inserted opposite the site of damage. Repair of these BER intermediates can be mediated by AP endonucleases, and the absence of AP endonucleases will significantly increase TM caused by BER intermediates.

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