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  • Transcription can also be impaired


    Transcription can also be impaired by lesions in the template DNA strand, which may lead to stalling of RNA polymerase (RNAP) or to transcriptional mutagenesis, thus producing mutant RNAs and proteins [[3], [4], [5], [6], [7]]. At the same time, RNAP can act as a sensor for DNA lesions, by attracting the DNA repair machinery to the damaged template sites during transcription coupled repair (TCR) (reviewed in Ref. [8]). Furthermore, stalled transcription complexes can severely compromise genome stability by colliding with the replication machinery [9,10]. Bacterial dihydrofolate reductase inhibitor antibacterial contain a single RNAP, and the process of DNA damage recognition and repair should be highly coordinated with transcription to allow efficient gene expression and DNA replication. However, the ability of bacterial RNAP to transcribe damaged DNA templates has not been systematically studied. Only a handful of lesions have been analyzed in the bacterial transcription system in vitro. In particular, it was shown that similarly to DNA polymerases, the abasic site and 8-oxoguanine promote mutagenic insertion of ATP in the RNA transcript, with transient RNAP pausing [[4], [5], [6]]. At the same time, the molecular mechanisms of translesion RNA synthesis remain poorly understood for most types of damaged nucleotides. In this work, we directly compared the effects on transcription by E. coli RNAP of several types of lesions commonly found in the genomic DNA: thymine dimer (CPD, cyclobutane pyrimidine dimer); 1,N6-ethenoadenine (εA); abasic site (AP); 8-oxoguanine (8oxoG), and thymine glycol (TG). We for the first time demonstrated that CPD and εA severely inhibit the activity of bacterial RNAP in vitro, allowing only very slow incorporation of ATP (the same specificity as observed for the AP-site), while TG has only a weak effect on transcription. Furthermore, we showed that 8oxoG is highly mutagenic because it enables not only insertion of a noncomplementary ATP but also its efficient extension by RNAP. Finally, we studied the effects of amino acid substitutions in the active site of E. coli RNAP on transcription of damaged DNA and identified mutations with altered transcription efficiency.
    Materials and methods
    Discussion CPD is the most common UV light-induced DNA lesion that severely inhibits DNA replication [2]. We demonstrated that CPD also strongly impairs transcription by bacterial RNAP, with very slow nucleotide insertion opposite both thymine dimer residues. Similarly to our observations, it was shown that nucleotide insertion opposite CPD by eukaryotic RNAP II is dramatically slowed down in comparison with undamaged templates [3,[16], [17], [18]]. This is likely explained by impaired RNAP translocation and may be accompanied by RNAP backtracking [18,19]. Furthermore, certain substitutions in the trigger loop of RNAP II were shown to stimulate translesion synthesis and cell survival after UV irradiation, possibly by promoting nucleotide insertion [19]. In contrast, our analyzed substitutions in the BH and switch2 of E. coli RNAP inhibited transcription. We revealed that another common lesion εA can completely block transcription, likely by disrupting complementary pairing with the incoming NTP. Although εA has never been studied with any RNAP, a similar guanine modification, 1,N2-ethenoguanine, could block RNA synthesis by eukaryotic RNAP II [20], suggesting that the effects of this type of modification are similar for bacterial and eukaryotic RNAPs. The AP-site was previously shown to induce transient RNAP pausing and ATP insertion by both E. coli and eukaryotic RNAPs, followed by further RNA extension [4,7]. For eukaryotic RNAP, the AP-site was recently demonstrated to slow down nucleotide incorporation both opposite and after the lesion [21]. We also observed pausing by bacterial RNAP before and after ATP insertion demonstrating that the mechanism of AP-site bypass is similar for these RNAPs.