Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis

Abstract

Maintaining high transcriptional fidelity is essential for life. Some DNA lesions lead to significant changes in transcriptional fidelity. In this review, we will summarize recent progress towards understanding the molecular basis of RNA polymerase II (Pol II) transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. In particular, we will focus on the three key checkpoint steps of controlling Pol II transcriptional fidelity: insertion (specific nucleotide selection and incorporation), extension (differentiation of RNA transcript extension of a matched over mismatched 3'-RNA terminus), and proofreading (preferential removal of misincorporated nucleotides from the 3'-RNA end). We will also discuss some novel insights into the molecular basis and chemical perspectives of controlling Pol II transcriptional fidelity through structural, computational, and chemical biology approaches.

Keywords: Chemical biology; Computational biology; DNA damage recognition; Fidelity control; Nonpolar isostere; RNA polymerase II; Structural biology; Synthetic nucleotide analogs; Transcription elongation; Transcriptional lesion bypass; Unlocked nucleic acid.

Figure 1

Schematic model showing the three fidelity checkpoint steps of Pol II transcription. The matched nucleotide (in green) is selected over an incorrect substrate (orange) during the incorporation step (step 1) and the RNA transcript can be efficiently elongated from a matched pair (step 2). In addition, proofreading is required to remove misincorporated nucleotides, backtrack, and reactivate the transcription process (step 3). The DNA template and RNA product are shown in blue and red, respectively. The active site of Pol II is shown in a dash box.

Figure 2

Structure of Pol II elongation complexes (EC). (a) Interaction networks between the binding NTP and Pol II EC in the active site (PDB ID: 2E2H). The incoming NTP enters the active site through the secondary channel of Pol II, as indicated with the blue arrow. (b) The nucleotide addition cycle. The NTP can first bind to the “entry site”, and rotates into the “addition site” to form the matched base pair with the DNA template nucleotide. After catalysis, the pre-translocation state will translocate one base-step forward to create a new active site and reinitiate the nucleotide addition cycle.

Figure 3

NTP recognition by the Pol II EC. (a) Interaction networks between the binding NTP and Pol II EC in the active site. The bridge helix (BH, in magenta), trigger loop (TL, in cyan), RNA/DNA hybrid chain (in yellow), NTP (in green), and the Pol II residues interacting with the NTP (in gray) are shown. (b) Schematic model of chemical interactions involved in the recognition interaction networks. The hydrogen bonds within the base pairs and the stacking interactions between the base groups in stabilizing the NTP are indicated as dashed grey lines. The highlighted TL residue Leu1081 serves as an anchor point to position the NTP in the active site. (c) The closing motion of the TL domain can stabilize the NTP in the active site and further facilitates the catalytic reaction. The various forms of the TL captured by X-ray studies are shown in different colors. (d) Substrate misincorporation can lead to Pol II EC backtracking (PDB ID: 3GTG).

Figure 4

A complete nucleotide addition cycle during the transcriptional elongation process, which primarily consists of the following steps: (1) The NTP (in orange) binds to the post-translocation state of the Pol II EC, accompanied by the TL closing motion. (2) The catalytic reaction takes place and one pyrophosphate ion (PP_i) is formed. (3) PP_i is released from the active site, followed by the TL opening motion. (4) The pretranslocation state of Pol II EC translocates to the post-translocation state to reinitiate another nucleotide addition cycle. (5) When misincorporation occurs, the Pol II EC can move in reverse direction to form a backtracked state. The NTP- and modeled PP_i-bound Pol II EC complexes are shown in the inset where the TL residues Leu1081 and His1085 are highlighted in cyan.

Figure 5

Chemical biology tools to dissect the contributions of hydrogen bonding and the sugar backbone to transcriptional fidelity. (a) Chemical structures of several thymidine (T) analogues (H, F, L, B, I) with sub-angstrom increments in size and varying abilities to form hydrogen bonds, compared to wild-type. (b) The unlocked nucleoside was designed to evaluate the roles of the sugar backbone in transcriptional fidelity.

Figure 6

8-oxo dG adopts an anti-form when pairing with cytosine and a syn-form when pairing with adenine in the Pol II active site.

Figure 7

The structural basis of the bifunctional bulky DNA lesion-containing Pol II ECs. (a) Stick model of the cyclobutane pyrimidine dimer (CPD, left panel) and the crystal structure of the stalled Pol II EC with one CPD lesion in the DNA template (right panel). (b) Stick model of the cisplatin-induced 1,2-dGpG intra-strand cross-link (left panel) and the crystal structure of the stalled Pol II EC with the 1,2-dGpG cisplatin DNA lesion in the DNA template (right panel).

Figure 8

Structural basis of the monofunctional bulky DNA lesion-containing Pol II EC. (a) Chemical structures of cisplatin, pyriplatin and phenanthriplatin. (b) The crystal structure of the Pol II EC stalled at a pyriplatin-DNA monofunctional adduct.