Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis
Introduction
Eukaryotic RNA polymerase II (Pol II) is one of the central enzymes for the first key step of gene expression [1]. During transcription, Pol II reads the DNA template and synthesizes a complementary RNA strand. The functional RNA molecules include precursors of protein-coding messenger RNAs as well as non-coding RNAs which may have important and diverse biological roles. Therefore, maintaining a highly accurate transfer of genetic information from DNA to RNA (high transcriptional fidelity) is essential for the process of life [2].
Pol II, as a highly specific “DNA reader” and “RNA writer”, is able to maintain a low error rate during transcription (less than 10−5) [3], [4]. The Pol II active site forms a network of interactions that specifically recognizes cognate nucleoside triphosphates and excludes non-cognate ones through three fidelity checkpoint steps [3], [5]. These three checkpoint steps include 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) (Fig. 1) [3], [5], [6].
RNA Pol II has also been proposed to be a highly selective DNA damage sensor, since it constantly scans the transcribed genome during transcription [7], [8]. In fact, a potentially lethal challenge that all cells and organisms must constantly face is the generation of harmful genomic DNA lesions caused by endogenous and environmental agents [9], [10]. There can be as many as one million DNA lesions generated in a cell per day [11]. Many of these lesions cause significant DNA structural and chemical alterations. The presence of DNA lesions within highly transcribed genomic regions significantly alters Pol II transcription with deleterious consequences [12], [13], [14], [15].
Biochemical and genetic studies have shown that the action Pol II takes when encountering DNA damage is lesion specific [7], [13], [14], [15], [16], [17]. Pol II can either bypass, backtrack, or stall at DNA lesions. Pol II transcriptional bypass may cause misincorporation within the RNA transcript, termed transcriptional mutagenesis [15], [18]. Pol II backtracking leads to intrinsic or transcription factor IIS (TFIIS)-mediated RNA transcript cleavage. Pol II stalling initiates a specialized DNA repair pathway, termed transcription-coupled repair (TCR) [13], [15], [16], [17]. The TCR pathway, first discovered by the Hanawalt Lab, specifically repairs DNA lesions in the transcribed genome [13], [16], [19], [20], [21], [22], [23]. In this pathway, the Cockayne Syndrome B protein (CSB) is one of the first proteins recruited to the arrested Pol II site and is involved in the early stages of TCR, although the detailed recruiting mechanism remains to be elucidated [22], [24], [25], [26], [27], [28]. Finally, as the last resort to remove persistently arrested Pol II, the arrested Pol II undergoes ubiquitylation and degradation [7], [14], [29]. Defects in genes involved in TCR cause premature aging and several human diseases, such as UV-sensitive syndrome, Cockayne Syndrome, Xeroderma Pigmentosum, Trichothiodystrophy, and Cerebro-Oculo-Facio-Skeletal Syndrome [13], [30], [31], [32], [33], [34], [35]. Several excellent reviews on transcription-coupled DNA damage processing can be found elsewhere [13], [14], [15], [16], [17]. In this review, we will focus on discussing some novel insights into the molecular basis and chemical perspectives in controlling Pol II transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis through structural, computational and chemical biology approaches.
Section snippets
Understanding the molecular basis of transcriptional fidelity through structural biology approaches
Pol II is an RNA polymerase containing 12 subunits with a total molecular weight of 550 kDa. A combination of genetic, biochemical, and structural studies have shed new light on the Pol II transcriptional mechanism at an atomic level over the last decade [5], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56]. There are several excellent reviews focusing on Pol II enzymatic catalysis and transcriptional regulation
Recent studies on DNA lesion-induced transcriptional mutagenesis
It has long been recognized that Pol II can bypass certain types of DNA lesions [15], [18], [116], [117], [118]. Among these lesions, some types of DNA damage frequently cause nucleotide misincorporation into an RNA transcript (error-prone transcription bypass) in a manner similar to translesion synthesis by error-prone DNA polymerases, while other types of DNA lesions have little effect on transcriptional fidelity (error-free transcription bypass) [15], [119], [120]. Error-prone transcription
Conclusions
In summary, recent advances in structural biology, computational biology and synthetic chemical biology provide us with new perspectives in studying the molecular basis of transcriptional fidelity and functional interplay between DNA lesions and Pol II transcription. We now have detailed molecular mechanisms of Pol II transcriptional fidelity maintenance and have started to gain important insights into what chemical interactions and intrinsic structural features of nucleic acid are important
Conflicts of Interest
There is no conflict of interest for all authors.
Acknowledgements
D.W. acknowledges the NIH (GM102362), Kimmel Scholars award from the Sidney Kimmel Foundation for Cancer Research, and start-up funds from Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD. E.T.K. acknowledges the NIH (GM072705 and GM068122) for support. S.W.P. acknowledges the UCSD Graduate Training Program in Cellular and Molecular Pharmacology training grant (T32 GM007752).
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