In this work we directly compared the effects on transcripti

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 pten pathway substitutions in the active site of E. coli RNAP on transcription of damaged DNA and identified mutations with altered transcription efficiency.
Materials and methods
Results
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. Interestingly, all three lesions that significantly inhibited transcription – CPD, εA and the AP site, – promoted insertion of adenine residues opposite the damaged nucleotide. Thus, bacterial RNAP seems to follow the so-called ‘A-rule’ for incorporation of nucleotides opposite AP-sites and bulky lesions, similarly to many DNA polymerases [22]. ATP is a preferred nucleotide for the active site of both bacterial [23] and eukaryotic [21] RNAPs, which likely promotes nontemplated ATP insertion. Indeed, RNAP II was shown to insert A opposite CPD in a nontemplated manner [19]. Other bulky lesions may be transcribed by RNAP in a similar way. 8oxoG is the most common oxidative DNA lesion that has been studied in both eukaryotic and bacterial transcription systems. When located in the template DNA strand, it promotes misincorporation of adenine and causes weak transcriptional pausing by both bacterial and eukaryotic RNAPs [[5], [6], [7],24]. Misincorporation of ATP likely occurs due to the formation of a Hoogsteen base-pair with 8oxoG bound in the syn conformation [25]. We confirmed that bacterial RNAP can insert both CTP and ATP opposite the lesion and also demonstrated that adenine-containing transcripts are preferably elongated after misincorporation, likely because of a more favorable conformation of the noncanonical 8oxoG-A pair during translocation of the RNA-DNA hybrid within RNAP. In agreement with this, the major pause on the 8oxoG template was observed downstream of the lesion (Fig. 2). Thus, the misincorporated adenine can be preferably extended on the 8oxoG templates both in vitro and in vivo resulting in transcriptional mutagenesis [5,25].