TRANSCRIPTION 2016, VOL. 7, NO. 3, 57–62 http://dx.doi.org/10.1080/21541264.2016.1168506
POINT-OF-VIEW
RNA polymerase II acts as a selective sensor for DNA lesions and endogenous DNA modifications Ji Hyun Shiny, Liang Xuy, and Dong Wang Department of Cellular and Molecular Medicine, School of Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA, USA
ABSTRACT
ARTICLE HISTORY
During transcription elongation, RNA polymerase II (pol II) travels along the DNA template across thousands to millions of nucleotides and accurately synthesizes the complementary RNA transcripts. Apart from its canonical function as a key enzyme for DNA-dependent RNA synthesis, pol II also functions as a selective sensor to recognize DNA lesions or epigenetic modifications.
Received 3 February 2016 Revised 11 March 2016 Accepted 14 March 2016 KEYWORDS
DNA lesions; DNA modifications; epi-DNA recognition loop; RNA polymerase II; sensor
RNA pol II is a key enzyme responsible for the first step of gene expression. During transcription, pol II reads along the template DNA strand and incorporates the matched nucleotide substrate for RNA transcripts synthesis. Maintenance of high pol II transcriptional fidelity is critical for fundamental biological processes and transcriptional errors contribute to aging and human diseases.1,2 At the transcription elongation phase, pol II transcriptional fidelity is maintained via at least three fidelity checkpoint steps: the nucleotide insertion step, the RNA transcript extension step, and the proofreading step.3 Several important motifs of pol II have been identified that contribute to transcriptional fidelity control. For example, the trigger loop (TL) of the Rpb 1 sub-unit is a highly conserved domain in various multi-subunit RNA polymerases that is responsible for positioning the substrate, rapid catalysis of phosphodiester bond formation, and substrate selection.4-7 The TL undergoes a conformational change from an open, inactive state to a closed, active state in the presence of a matched NTP substrate, to seal off the active site and position the substrate to be poised for catalysis.4 In addition, Rbp9, a small conserved subunit of pol II, also plays an important role in transcriptional
proofreading.8 Furthermore, we also recently systematically examined the individual contributions of chemical interactions (such as hydrogen bonds and base stacking) and nucleic acid structural motifs in Pol II transcriptional fidelity control.3,9,10 The genomic DNA is constantly under attack by environmental stress (such as UV radiation or tobacco smoke) and subject to oxidative damage from byproducts of metabolism (such as free radicals and reactive species). During transcription elongation, pol II may encounter various DNA modifications or lesions while transcribing along the DNA template (Fig. 1). These DNA modifications and lesions may lead to different outcomes depending on their chemical nature, which may lead to transcriptional mutagenesis, transcriptional arrest, transcription coupled repair pathway, or pol II ubiquitylation.11-14 In all of these cases, pol II is proposed to function as a specific sensor to sense DNA modifications and lesions through specific interactions with the pol II active site.15 Over the last few years, significant new structural insights were obtained in the understanding of how pol II interacts with different types of DNA lesions at the active site. For example, UV-induced cyclobutane pyrimidine dimer (CPD) lesions (Fig. 1) significantly
CONTACT Dong Wang [email protected] Department of Cellular and Molecular Medicine, School of Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ktrn. y These two authors contributed equally to this work. © 2016 Taylor & Francis Group, LLC
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Figure 1. During transcription, RNA polymerase II encounters a variety of DNA modifications including DNA lesions (8-oxo-G, CPD and CydA as examples, boxed in blue) and enzyme-catalyzed endogenous DNA modifications (modifications of cytosine and thymine as examples, boxed in green).
distort the DNA template structure and strongly inhibit pol II transcription elongation.12,16 The presence of DNA lesions caused pol II to significantly slow down the template-dependent substrate incorporation step as well as the extension step.12 Interestingly, another structurally unrelated monofunctional bulky DNA lesion arising from oxidative damage, cyclopurines (CydA) (Fig. 1), also similarly inhibit pol II transcription by reducing the rate of substrate incorporation and extension.17 Furthermore, both DNA lesions cause pol II to preferentially incorporate AMP opposite to damaged DNA bases in a non-template manner, according to the so-called “A rule”.18 These phenomena raise the question of how pol II recognizes and processes these structurally distinct DNA lesions in a similar pattern. Two recent pol II elongation complex structures containing either CPD or CydA lesions shed new structural insights into answering this question.12,17 Strikingly, despite significant structural differences between CPD and CydA DNA lesions, it was found that both DNA lesions are accommodated at a similar location at the pol II active site. Both DNA lesions are stuck above the bridge helix and arrested at essentially a “half-way” position of template translocation from the downstream canonical iC2 position to iC1 position.12,17 Indeed, the metastable states in which the template base
partially crosses over the bridge helix were also identified during normal pol II translocation on non-damaged DNA templates using molecular dynamic simulation.19 These translocation intermediate states are rate-limiting steps for translocation and require significant conformational changes for the DNA template (switching from canonical B-form DNA duplex at iC2 site to rotate roughly a-90 degree to crossover bridge helix to realign with upstream DNA template). The common DNA lesion arrest sites may therefore indicate that such a crossover step is a major checkpoint for pol II to examine if there are any DNA lesions that compromise DNA structural integrity and backbone flexibility. One, of course, needs to take note that some DNA lesions can skip such a checkpoint step and deliver the DNA lesions to the iC1 position and does not significantly inhibit transcription elongation.11 For example, pol II can bypass 8-Oxo-20 -deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, without arrest at the DNA lesions. The presence of the 8-carbonyl group of 8-oxo-dG destabilizes the anti-conformation for cytosine incorporation and favors the syn-conformation for template-dependent mismatched AMP incorporation.20 Besides DNA lesions that threaten genome integrity, there are other groups of DNA modifications that are catalyzed by enzymes and have regulatory roles in
TRANSCRIPTION
gene expression (Fig. 1). For example, DNA methyltransferases (DNMTs) can methylate cytosine to generate 5-methylcytosine (5 mC). 5 mC is the most common DNA modification, often enriched at enhancer and promoter regions, and plays an important role in regulating gene transcription, and particularly functions as a repressive epigenetic mark.21 5 mC can also undergo demethylation catalyzed by a combination of a series of enzymes. Ten eleven translocation (Tet) proteins can oxidize mCs (methylcytosines) in a step-wise manner and generate oxidized methylcytosine (oxi-mC) intermediates, 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC), and 5carboxylcytosine (5caC) (Fig. 1). 5fC and 5caC can be recognized and removed by thymine DNA glycosylase (TDG) and subsequently repaired to regenerate the unmodified cytosine via the base excision pathway.22 Recent evidence suggests that these oxidized 5mCs (oxi-mCs) are stable DNA modifications and may have functional roles beyond just being a demethylation intermediate.23 Indeed, oxi-mCs can be recognized by a set of protein complexes that are involved in transcription and chromatin regulation and the DNA repair process, such as Swi/Snf remodeling complex sub-unit BAF170 (as 5caC reader), p53 (as 5fC readers), Mpg, and Neil3 (as 5hmC readers).24 To investigate whether pol II has the capability to discriminate these types of DNA modifications, we performed in vitro transcription assays and revealed that 5fC/5caC can induce transient transcriptional pausing in vitro.25 Further comparison of GRO-seq results of transcription elongation of WT (wild type) and TDG-KO (thymine DNA glycosylase – knock out) cell lines also support a negative correlation of the pol II elongation rate and endogenous 5fC/5caC levels, which is in good agreement with in vitro transcription results.26 To further investigate the structural basis of how pol II recognizes oxi-mCs, we solved the structure of a pol II elongation complex containing a 5caC at the iC1 site (Fig. 2A). The structure revealed that a major portion of 5caC is accommodated above the bridge helix (Fig. 2B), a similar location for the bulky CydA and CPD lesions. In contrast to the cases of CydA and CPD lesions enforced by covalent crosslinks to form a strong steric barrier for crossover of the bridge helix, we found a highly specific interaction between pol II and the 5caC, consisting of hydrogen bonds between the 5-
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carboxyl group of 5caC and the side chain of residue Q531 of the fork region of the Rpb2 subunit of pol II (Fig. 2C).26 We renamed this loop as the epi-DNA recognition loop for it can recognize the modified cytosine in the major groove of the template strand. These specific hydrogen bonds cause misalignment of the 5caC template and subsequently disrupt proper alignment of the GTP substrate and 30 -RNA terminus, and result in a partially open conformation of the TL. As a result, the GTP incorporation efficiency opposite 5caC is reduced. To further validate the functional roles of the Q:5caC interaction on GTP incorporation, two pol II point mutants, Rpb2 Q531H and Q531A, were purified and tested for in vitro GTP incorporation. Indeed, as expected, the Q531A mutant lost the ability to form the hydrogen bond and gained a significant increase in GTP incorporation specificity, while the Q531H mutant behaved like WT pol II, due to the formation of the hydrogen bond between the His residue and 5caC. Like 5caC, 5fC may form the similar hydrogen bond interaction with pol II through its carbonyl group as revealed by our modeling study.26 Apart from the well-known function that Pol II acts as a key enzyme for synthesizing protein-coding and non-coding RNA transcripts from non-damaged templates, emerging evidence reveals that pol II may have additional functional roles. Pol II can serve as a specific sensor for DNA lesion attack and signal for a variety of DNA damage response pathways. More importantly, here we propose that pol II can also work as a specific sensor for enzyme-catalyzed DNA modifications for regulatory functional roles. Pol II is able to recognize 5caC via a specific hydrogen bond interaction between the carboxyl group of 5caC and a Pol II residue and slow down pol II transcription elongation.26 It is expected that 5fC and 5fU may have similar interactions with pol II (Fig. 1). The functional interplay between Pol II and endogenous DNA modifications may be an emerging theme. Indeed, as another example, some thymine bases in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield Base J (b-D-glucosylhydroxymethyluracil) (Fig. 1) and Base J is reported to be required for proper transcription termination in Leishmania.27,28 The loss of internal Base J is accompanied by massive read through at RNA polymerase II termination sites. It would be interesting to further
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Figure 2. RNA polymerase II recognizes 5caC in an “above the bridge helix” position via the epi-DNA recognition loop (Epi-loop). (A) The overall core pol II elongation complex structure containing a sites-specific 5caC at iC1 site (Rpb2 is omitted). (B) Two 5caC conformers are observed in the pol II active site. Part of the bridge helix (BH) is highlighted in green. The nucleotide addition site is represented by a dotted oval. (C) The “midway” 5caC interacts with the Rpb2 Q531 residue via hydrogen bonds (black dotted lines). The epi-DNA recognition loop is shown in cyan.
explore the impact of other enzyme-catalyzed DNA modifications on transcription in the near future. Recent studies also shed new insights onto the pol II translocation process, and pausing and arrest during transcription elongation. For normal pol II translocation on non-damaged DNA templates, the crossover of the bridge helix is usually fast and translocation intermediates states are short-lived.19 The rapid translocation dynamics can be significantly slowed down by a variety of factors including specific DNA pausing sequences, DNA modifications or lesions, and a-amanitin. Aligning the structures of 5caC-paused pol II elongation complex with several recent bulky DNA lesionarrested complexes reveals new insights into pol II pausing and arrest upon DNA modifications/ lesions.12,17,26,29 Intriguingly, the iC1 transition template base in these 5caC-paused or bulky DNA lesion-arrested structures are all accommodated “above the bridge helix,” representing a common translocation checkpoint for a variety of endogenous DNA modifications and DNA lesions.12,17,26,29 Intriguingly, the similar “above the bridge helix” translocation intermediates are also observed for non-damaged pausing and arrest complexes revealed by the structures of the a-amanitin-
arrested pol II complex5,30 and the E. Coli RNAP pausing complex.31 Another interesting example is revealed by a recent structure of the RNA pol III elongation complex,32 in which the undamaged template iC1 nucleobase adopts a similar “above the bridge helix” position. Strikingly, this undamaged nucleobase is held at the “above the bridge helix” position through a specific hydrogen-bonding interaction with the pol III residue E506, the counterpart to the pol II Rpb2 Q531 residue that directly senses 5caC/5fC through specific hydrogenbonding interactions. Finally, there is a remarkable mechanistic similarity in 5caC recognition by several unrelated family proteins, which implicate a shared 5caC recognition mode.26 Intriguingly, for example, the residue Q369 in Wilms tumor (WT1) protein33 and the residue N157 in human TDG34 are both functionally equivalent to Q/H531 residues in pol II in recognizing the 5caC carboxyl group via specific hydrogen bonds. Another example is the zinc finger protein Zfp57. The WT Zfp57 doesn’t recognize 5caC; however, the Zfp57 E182Q is gain-of-function mutant that can selectively recognize 5caC via a similar recognition mode.35 Taken together, the data led us to speculate that 5caC could be a potential epigenetic mark for recognition
TRANSCRIPTION
by a variety of “protein readers” via specific hydrogenbonding interactions with its 5-carboxyl moiety. Conclusively, as RNA pol II transcribes along the DNA template and synthesizes RNA molecules, its ability to sense a variety of DNA damage and modifications may add another regulatory layer which allows pol II to respond to DNA damage stress as well as other regulatory cues. Such transcriptional responses can include lesion bypass, transcriptional pausing or arrest, recruitment of a variety of protein complexes, such as the chromatin remodeling complex, histone modifying enzymes, co-transcriptional RNA processing machineries, or DNA repair machinery.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding D.W. acknowledges the National Institutes of Health (GM102362).
References [1] Bregeon D, Doetsch PW. Transcriptional mutagenesis: causes and involvement in tumour development. Nat Rev Cancer 2011; 11(3):218-227 [2] Vermulst M, Denney AS, Lang MJ, Hung CW, Moore S, Moseley MA, Thompson JW, Madden V, Gauer J, Wolfe KJ et al. Transcription errors induce proteotoxic stress and shorten cellular lifespan. Nat Commun 2015; 6:8065 [3] Xu L, Wang W, Chong J, Shin JH, Xu J, Wang D. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications. Crit Rev Biochem Mol Biol 2015; 50(6):503-519 [4] Wang D, Bushnell DA, Westover KD, Kaplan CD, Kornberg RD. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis. Cell 2006; 127(5):941-954 [5] Kaplan CD, Larsson K-M, Kornberg RD. The RNA polymerase II trigger loop functions in substrate selection and is directly targeted by a-amanitin. Mol Cell 2008; 30 (5):547-556 [6] Huang X, Wang D, Weiss DR, Bushnell DA, Kornberg RD, Levitt M. RNA polymerase II trigger loop residues stabilize and position the incoming nucleotide triphosphate in transcription. Proc Natl Acad Sci U S A 2010; 107(36):15745-15750 [7] Kireeva ML, Nedialkov YA, Cremona GH, Purtov YA, Lubkowska L, Malagon F, Burton ZF, Strathern JN, Kashlev M. Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation. Mol Cell 2008; 30(5):557-566
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[8] Walmacq C, Kireeva ML, Irvin J, Nedialkov Y, Lubkowska L, Malagon F, Strathern JN, Kashlev M. Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II. J Biol Chem 2009; 284 (29):19601-19612 [9] Zhang S, Wang D. Understanding the Molecular Basis of RNA Polymerase II Transcription. Isr J Chem 2013; 53:442-449 [10] Xu L, Da L, Plouffe SW, Chong J, Kool E, Wang D. Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis. DNA Repair (Amst) 2014; 19:71-83 [11] Scicchitano DA, Olesnicky EC, Dimitri A. Transcription and DNA adducts: what happens when the message gets cut off? DNA Repair (Amst) 2004; 3(12):1537-1548 [12] Walmacq C, Cheung AC, Kireeva ML, Lubkowska L, Ye C, Gotte D, Strathern JN, Carell T, Cramer P, Kashlev M. Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage. Mol Cell 2012; 46(1):18-29 [13] Hanawalt PC, Spivak G. Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol 2008; 9(12):958-970 [14] Wilson MD, Harreman M, Svejstrup JQ. Ubiquitylation and degradation of elongating RNA polymerase II: the last resort. Biochim Biophys Acta 2013; 1829(1):151-157 [15] Lindsey-Boltz LA, Sancar A. RNA polymerase: the most specific damage recognition protein in cellular responses to DNA damage? Proc Natl Acad Sci U S A 2007; 104 (33):13213-13214 [16] Brueckner F, Hennecke U, Carell T, Cramer P. CPD damage recognition by transcribing RNA polymerase II. Science 2007; 315(5813):859-862 [17] Walmacq C, Wang L, Chong J, Scibelli K, Lubkowska L, Gnatt A, Brooks PJ, Wang D, Kashlev M. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions. Proc Natl Acad Sci U S A 2015; 112(5):E410-419 [18] Obeid S, Blatter N, Kranaster R, Schnur A, Diederichs K, Welte W, Marx A. Replication through an abasic DNA lesion: structural basis for adenine selectivity. EMBO J 2010; 29(10):1738-1747 [19] Silva DA, Weiss DR, Pardo Avila F, Da LT, Levitt M, Wang D, Huang X. Millisecond dynamics of RNA polymerase II translocation at atomic resolution. Proc Natl Acad Sci U S A 2014; 111(21):7665-7670 [20] Damsma GE, Cramer P. Molecular basis of transcriptional mutagenesis at 8-oxoguanine. J Biol Chem 2009; 284(46):31658-31663 [21] Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science 2001; 293(5532):10681070 [22] Wu H, Zhang Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell 2014; 156 (1–2):45-68 [23] Bachman M, Uribe-Lewis S, Yang X, Burgess HE, Iurlaro M, Reik W, Murrell A, Balasubramanian S. Five-
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Formylcytosine can be a stable DNA modification in mammals. Nat Chem Biol 2015; 11(8):555-557 Spruijt CG, Gnerlich F, Smits AH, Pfaffeneder T, Jansen PW, Bauer C, M€ unzel M, Wagner M, M€ uller M, Khan F et al. Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives. Cell 2013; 152(5):1146-1159 Kellinger MW, Song CX, Chong J, Lu XY, He C, Wang D. Five-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nat Struct Mol Biol 2012; 19(8):831-833 Wang L, Zhou Y, Xu L, Xiao R, Lu X, Chen L, Chong J, Li H, He C, Fu XD et al. Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex. Nature 2015; 523(7562):621-625 Huang Y, Rao A. New functions for DNA modifications by TET-JBP. Nat Struct Mol Biol 2012; 19 (11):1061-1064 van Luenen HG, Farris C, Jan S, Genest PA, Tripathi P, Velds A, Kerkhoven RM, Nieuwland M, Haydock A, Ramasamy G et al. Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania. Cell 2012; 150(5):909-921 Wang D, Zhu G, Huang X, Lippard SJ. X-ray structure and mechanism of RNA polymerase II stalled at an
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antineoplastic monofunctional platinum-DNA adduct. Proc Natl Acad Sci U S A 2010; 107(21):9584-9589 Brueckner F, Cramer P. Structural basis of transcription inhibition by a-amanitin and implications for RNA polymerase II translocation. Nat Struct Mol Biol 2008; 15 (8):811-818 Weixlbaumer A, Leon K, Landick R, Darst SA. Structural basis of transcriptional pausing in bacteria. Cell 2013; 152(3):431-441 Hoffmann NA, Jakobi AJ, Moreno-Morcillo M, Glatt S, Kosinski J, Hagen WJ, Sachse C, M€ uller CW. Molecular structures of unbound and transcribing RNA polymerase III. Nature 2015; 528(7581):231-236 Hashimoto H, Olanrewaju YO, Zheng Y, Wilson GG, Zhang X, Cheng X. Wilms tumor protein recognizes 5carboxylcytosine within a specific DNA sequence. Genes Dev 2014; 28(20):2304-2313 Zhang L, Lu X, Lu J, Liang H, Dai Q, Xu GL, Luo C, Jiang H, He C. Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA. Nat Chem Biol 2012; 8(4):328-330 Liu Y, Olanrewaju YO, Zhang X, Cheng X. DNA recognition of 5-carboxylcytosine by a Zfp57 mutant at an atomic resolution of 0.97 A. Biochemistry 2013; 52(51):9310-9317
POINT-OF-VIEW
RNA polymerase II acts as a selective sensor for DNA lesions and endogenous DNA modifications Ji Hyun Shiny, Liang Xuy, and Dong Wang Department of Cellular and Molecular Medicine, School of Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA, USA
ABSTRACT
ARTICLE HISTORY
During transcription elongation, RNA polymerase II (pol II) travels along the DNA template across thousands to millions of nucleotides and accurately synthesizes the complementary RNA transcripts. Apart from its canonical function as a key enzyme for DNA-dependent RNA synthesis, pol II also functions as a selective sensor to recognize DNA lesions or epigenetic modifications.
Received 3 February 2016 Revised 11 March 2016 Accepted 14 March 2016 KEYWORDS
DNA lesions; DNA modifications; epi-DNA recognition loop; RNA polymerase II; sensor
RNA pol II is a key enzyme responsible for the first step of gene expression. During transcription, pol II reads along the template DNA strand and incorporates the matched nucleotide substrate for RNA transcripts synthesis. Maintenance of high pol II transcriptional fidelity is critical for fundamental biological processes and transcriptional errors contribute to aging and human diseases.1,2 At the transcription elongation phase, pol II transcriptional fidelity is maintained via at least three fidelity checkpoint steps: the nucleotide insertion step, the RNA transcript extension step, and the proofreading step.3 Several important motifs of pol II have been identified that contribute to transcriptional fidelity control. For example, the trigger loop (TL) of the Rpb 1 sub-unit is a highly conserved domain in various multi-subunit RNA polymerases that is responsible for positioning the substrate, rapid catalysis of phosphodiester bond formation, and substrate selection.4-7 The TL undergoes a conformational change from an open, inactive state to a closed, active state in the presence of a matched NTP substrate, to seal off the active site and position the substrate to be poised for catalysis.4 In addition, Rbp9, a small conserved subunit of pol II, also plays an important role in transcriptional
proofreading.8 Furthermore, we also recently systematically examined the individual contributions of chemical interactions (such as hydrogen bonds and base stacking) and nucleic acid structural motifs in Pol II transcriptional fidelity control.3,9,10 The genomic DNA is constantly under attack by environmental stress (such as UV radiation or tobacco smoke) and subject to oxidative damage from byproducts of metabolism (such as free radicals and reactive species). During transcription elongation, pol II may encounter various DNA modifications or lesions while transcribing along the DNA template (Fig. 1). These DNA modifications and lesions may lead to different outcomes depending on their chemical nature, which may lead to transcriptional mutagenesis, transcriptional arrest, transcription coupled repair pathway, or pol II ubiquitylation.11-14 In all of these cases, pol II is proposed to function as a specific sensor to sense DNA modifications and lesions through specific interactions with the pol II active site.15 Over the last few years, significant new structural insights were obtained in the understanding of how pol II interacts with different types of DNA lesions at the active site. For example, UV-induced cyclobutane pyrimidine dimer (CPD) lesions (Fig. 1) significantly
CONTACT Dong Wang [email protected] Department of Cellular and Molecular Medicine, School of Medicine, Skaggs School of Pharmacy and Pharmaceutical Sciences, The University of California, San Diego, La Jolla, CA 92093-0625, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ktrn. y These two authors contributed equally to this work. © 2016 Taylor & Francis Group, LLC
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Figure 1. During transcription, RNA polymerase II encounters a variety of DNA modifications including DNA lesions (8-oxo-G, CPD and CydA as examples, boxed in blue) and enzyme-catalyzed endogenous DNA modifications (modifications of cytosine and thymine as examples, boxed in green).
distort the DNA template structure and strongly inhibit pol II transcription elongation.12,16 The presence of DNA lesions caused pol II to significantly slow down the template-dependent substrate incorporation step as well as the extension step.12 Interestingly, another structurally unrelated monofunctional bulky DNA lesion arising from oxidative damage, cyclopurines (CydA) (Fig. 1), also similarly inhibit pol II transcription by reducing the rate of substrate incorporation and extension.17 Furthermore, both DNA lesions cause pol II to preferentially incorporate AMP opposite to damaged DNA bases in a non-template manner, according to the so-called “A rule”.18 These phenomena raise the question of how pol II recognizes and processes these structurally distinct DNA lesions in a similar pattern. Two recent pol II elongation complex structures containing either CPD or CydA lesions shed new structural insights into answering this question.12,17 Strikingly, despite significant structural differences between CPD and CydA DNA lesions, it was found that both DNA lesions are accommodated at a similar location at the pol II active site. Both DNA lesions are stuck above the bridge helix and arrested at essentially a “half-way” position of template translocation from the downstream canonical iC2 position to iC1 position.12,17 Indeed, the metastable states in which the template base
partially crosses over the bridge helix were also identified during normal pol II translocation on non-damaged DNA templates using molecular dynamic simulation.19 These translocation intermediate states are rate-limiting steps for translocation and require significant conformational changes for the DNA template (switching from canonical B-form DNA duplex at iC2 site to rotate roughly a-90 degree to crossover bridge helix to realign with upstream DNA template). The common DNA lesion arrest sites may therefore indicate that such a crossover step is a major checkpoint for pol II to examine if there are any DNA lesions that compromise DNA structural integrity and backbone flexibility. One, of course, needs to take note that some DNA lesions can skip such a checkpoint step and deliver the DNA lesions to the iC1 position and does not significantly inhibit transcription elongation.11 For example, pol II can bypass 8-Oxo-20 -deoxyguanosine (8-oxo-dG), a common oxidative DNA lesion, without arrest at the DNA lesions. The presence of the 8-carbonyl group of 8-oxo-dG destabilizes the anti-conformation for cytosine incorporation and favors the syn-conformation for template-dependent mismatched AMP incorporation.20 Besides DNA lesions that threaten genome integrity, there are other groups of DNA modifications that are catalyzed by enzymes and have regulatory roles in
TRANSCRIPTION
gene expression (Fig. 1). For example, DNA methyltransferases (DNMTs) can methylate cytosine to generate 5-methylcytosine (5 mC). 5 mC is the most common DNA modification, often enriched at enhancer and promoter regions, and plays an important role in regulating gene transcription, and particularly functions as a repressive epigenetic mark.21 5 mC can also undergo demethylation catalyzed by a combination of a series of enzymes. Ten eleven translocation (Tet) proteins can oxidize mCs (methylcytosines) in a step-wise manner and generate oxidized methylcytosine (oxi-mC) intermediates, 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC), and 5carboxylcytosine (5caC) (Fig. 1). 5fC and 5caC can be recognized and removed by thymine DNA glycosylase (TDG) and subsequently repaired to regenerate the unmodified cytosine via the base excision pathway.22 Recent evidence suggests that these oxidized 5mCs (oxi-mCs) are stable DNA modifications and may have functional roles beyond just being a demethylation intermediate.23 Indeed, oxi-mCs can be recognized by a set of protein complexes that are involved in transcription and chromatin regulation and the DNA repair process, such as Swi/Snf remodeling complex sub-unit BAF170 (as 5caC reader), p53 (as 5fC readers), Mpg, and Neil3 (as 5hmC readers).24 To investigate whether pol II has the capability to discriminate these types of DNA modifications, we performed in vitro transcription assays and revealed that 5fC/5caC can induce transient transcriptional pausing in vitro.25 Further comparison of GRO-seq results of transcription elongation of WT (wild type) and TDG-KO (thymine DNA glycosylase – knock out) cell lines also support a negative correlation of the pol II elongation rate and endogenous 5fC/5caC levels, which is in good agreement with in vitro transcription results.26 To further investigate the structural basis of how pol II recognizes oxi-mCs, we solved the structure of a pol II elongation complex containing a 5caC at the iC1 site (Fig. 2A). The structure revealed that a major portion of 5caC is accommodated above the bridge helix (Fig. 2B), a similar location for the bulky CydA and CPD lesions. In contrast to the cases of CydA and CPD lesions enforced by covalent crosslinks to form a strong steric barrier for crossover of the bridge helix, we found a highly specific interaction between pol II and the 5caC, consisting of hydrogen bonds between the 5-
59
carboxyl group of 5caC and the side chain of residue Q531 of the fork region of the Rpb2 subunit of pol II (Fig. 2C).26 We renamed this loop as the epi-DNA recognition loop for it can recognize the modified cytosine in the major groove of the template strand. These specific hydrogen bonds cause misalignment of the 5caC template and subsequently disrupt proper alignment of the GTP substrate and 30 -RNA terminus, and result in a partially open conformation of the TL. As a result, the GTP incorporation efficiency opposite 5caC is reduced. To further validate the functional roles of the Q:5caC interaction on GTP incorporation, two pol II point mutants, Rpb2 Q531H and Q531A, were purified and tested for in vitro GTP incorporation. Indeed, as expected, the Q531A mutant lost the ability to form the hydrogen bond and gained a significant increase in GTP incorporation specificity, while the Q531H mutant behaved like WT pol II, due to the formation of the hydrogen bond between the His residue and 5caC. Like 5caC, 5fC may form the similar hydrogen bond interaction with pol II through its carbonyl group as revealed by our modeling study.26 Apart from the well-known function that Pol II acts as a key enzyme for synthesizing protein-coding and non-coding RNA transcripts from non-damaged templates, emerging evidence reveals that pol II may have additional functional roles. Pol II can serve as a specific sensor for DNA lesion attack and signal for a variety of DNA damage response pathways. More importantly, here we propose that pol II can also work as a specific sensor for enzyme-catalyzed DNA modifications for regulatory functional roles. Pol II is able to recognize 5caC via a specific hydrogen bond interaction between the carboxyl group of 5caC and a Pol II residue and slow down pol II transcription elongation.26 It is expected that 5fC and 5fU may have similar interactions with pol II (Fig. 1). The functional interplay between Pol II and endogenous DNA modifications may be an emerging theme. Indeed, as another example, some thymine bases in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield Base J (b-D-glucosylhydroxymethyluracil) (Fig. 1) and Base J is reported to be required for proper transcription termination in Leishmania.27,28 The loss of internal Base J is accompanied by massive read through at RNA polymerase II termination sites. It would be interesting to further
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Figure 2. RNA polymerase II recognizes 5caC in an “above the bridge helix” position via the epi-DNA recognition loop (Epi-loop). (A) The overall core pol II elongation complex structure containing a sites-specific 5caC at iC1 site (Rpb2 is omitted). (B) Two 5caC conformers are observed in the pol II active site. Part of the bridge helix (BH) is highlighted in green. The nucleotide addition site is represented by a dotted oval. (C) The “midway” 5caC interacts with the Rpb2 Q531 residue via hydrogen bonds (black dotted lines). The epi-DNA recognition loop is shown in cyan.
explore the impact of other enzyme-catalyzed DNA modifications on transcription in the near future. Recent studies also shed new insights onto the pol II translocation process, and pausing and arrest during transcription elongation. For normal pol II translocation on non-damaged DNA templates, the crossover of the bridge helix is usually fast and translocation intermediates states are short-lived.19 The rapid translocation dynamics can be significantly slowed down by a variety of factors including specific DNA pausing sequences, DNA modifications or lesions, and a-amanitin. Aligning the structures of 5caC-paused pol II elongation complex with several recent bulky DNA lesionarrested complexes reveals new insights into pol II pausing and arrest upon DNA modifications/ lesions.12,17,26,29 Intriguingly, the iC1 transition template base in these 5caC-paused or bulky DNA lesion-arrested structures are all accommodated “above the bridge helix,” representing a common translocation checkpoint for a variety of endogenous DNA modifications and DNA lesions.12,17,26,29 Intriguingly, the similar “above the bridge helix” translocation intermediates are also observed for non-damaged pausing and arrest complexes revealed by the structures of the a-amanitin-
arrested pol II complex5,30 and the E. Coli RNAP pausing complex.31 Another interesting example is revealed by a recent structure of the RNA pol III elongation complex,32 in which the undamaged template iC1 nucleobase adopts a similar “above the bridge helix” position. Strikingly, this undamaged nucleobase is held at the “above the bridge helix” position through a specific hydrogen-bonding interaction with the pol III residue E506, the counterpart to the pol II Rpb2 Q531 residue that directly senses 5caC/5fC through specific hydrogenbonding interactions. Finally, there is a remarkable mechanistic similarity in 5caC recognition by several unrelated family proteins, which implicate a shared 5caC recognition mode.26 Intriguingly, for example, the residue Q369 in Wilms tumor (WT1) protein33 and the residue N157 in human TDG34 are both functionally equivalent to Q/H531 residues in pol II in recognizing the 5caC carboxyl group via specific hydrogen bonds. Another example is the zinc finger protein Zfp57. The WT Zfp57 doesn’t recognize 5caC; however, the Zfp57 E182Q is gain-of-function mutant that can selectively recognize 5caC via a similar recognition mode.35 Taken together, the data led us to speculate that 5caC could be a potential epigenetic mark for recognition
TRANSCRIPTION
by a variety of “protein readers” via specific hydrogenbonding interactions with its 5-carboxyl moiety. Conclusively, as RNA pol II transcribes along the DNA template and synthesizes RNA molecules, its ability to sense a variety of DNA damage and modifications may add another regulatory layer which allows pol II to respond to DNA damage stress as well as other regulatory cues. Such transcriptional responses can include lesion bypass, transcriptional pausing or arrest, recruitment of a variety of protein complexes, such as the chromatin remodeling complex, histone modifying enzymes, co-transcriptional RNA processing machineries, or DNA repair machinery.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
Funding D.W. acknowledges the National Institutes of Health (GM102362).
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