Interestingly all three lesions that significantly

Interestingly, all three lesions that significantly inhibited transcription – CPD, εA and the AP site, – promoted insertion of NS3694 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]. Thymine glycol severely inhibits DNA synthesis by replicative DNA polymerases and requires the action of specialized DNA polymerases for translesion synthesis (e.g. Ref. [26]). Eukaryotic and archaeal RNAPs were shown to pause after nucleotide insertion opposite TG [27,28]. Surprisingly, we found that transcription by bacterial RNAP is only slightly affected by this modification, probably because the RNAP active site is more tolerant to the duplex distortion caused by the nonplanar TG configuration, suggesting that this lesion may not be a subject for TCR. Finally, we analyzed several substitutions of amino acid residues in the RNAP active site involved in direct interactions with the transcribed DNA strand. Unexpectedly, some of these substitutions (R542A, Y795A and R798A) lacked any prominent effects on transcription of both normal and damaged DNA templates, despite affecting conserved RNAP residues involved in template interactions. At the same time, two substitutions, K334A in switch2 and T790 in the BH, inhibited transcription, and this effect was exacerbated on most damaged templates. The T790A substitution was previously shown to decrease RNAP activity in a number of assays, possibly by changing the BH conformation and/or its contacts with the template-NTP pair [29], likely explaining its effects on translesion synthesis. The effect of the K334A substitution may result from changes in RNAP contacts with the template DNA strand downstream of the active site and in the conformation of the clamp domain that holds the DNA-RNA duplex [14]. At the same time, the K334A substitution stimulated transcription past CPD, in contrast to other lesions, suggesting that it may help to overcome transcription stalling caused by this bulky lesion, possibly by loosening RNAP-DNA contacts near the active site.
Acknowledgements We thank I. Artsimovitch for plasmids and for the CPD template. This work was supported by the Grant of the Ministry of Higher Education and Science of the Russian Federation 14.W03.31.0007.
Introduction Cancer is the second cause of death and responsible for one-sixth of the deaths (estimated 9.6 million) all over the world in 2018 [1]. And the International Agency for Research on Cancer (IARC) estimated that there would be 21.7 million new cancer cases and 13 million cancer deaths by 2030 [2]. The development of new cancer treatments and drugs have been attracted great attention for decades. In 2017, FDA approved 12 oncology products which were 26% of the approved products [3].