Review pubs.acs.org/CR DNA Sequences That Interfere with Transcription: Implications for Genome Function and Stability † ‡ † Boris P. Belotserkovskii, Sergei M. Mirkin, and Philip C. Hanawalt*, † Department of Biology, Stanford University, Stanford, California 94305, United States ‡ Department of Biology, Tufts University, Medford, Massachusetts 02155, United States 1. INTRODUCTION The primary role of DNA-dependent RNA synthesis, or transcription, is to create components for the cellular machinery. The nascent RNA product of transcription is released from the DNA template and either serves as an intermediate message for protein synthesis or is used directly, as in the case of rRNA, tRNA, and various types of regulatory RNAs. Recently, a growing number of examples suggest that transcription per se, rather than its released product, could play a regulatory role in gene function or as a trigger for genomic modifications. The latter scenario is commonly attributed to anomalous progression of the RNA polymerase (RNAP), such as pausing CONTENTS or termination and/or retaining rather than releasing the nascent transcript. This has been implicated in class-switch 1. Introduction 8620 recombination and somatic hypermutation (reviewed in refs 1, − 2. Transcription Blockage Mechanisms 8621 2), telomere maintenance,3 and replication initiation.4 9 3. Effects of Unusual DNA Structures and DNA/RNA Anomalous transcription elongation has also been linked to Complexes on Transcription 8622 various deleterious phenomena, such as genomic instabilities, 3.1. General Introduction to DNA Structure and transcription−replication collisions, and transcription defi- DNA Supercoiling 8622 ciency in some hereditary human disorders (reviewed in refs 3.2. Overview of Unusual Structures and Their 10−14). Effects on Transcription 8623 Importantly, many of the DNA sequences at which RNAP 3.2.1. Branched/Looped Structures 8623 stumbles are prone to form alternative (non-B form) DNA 3.2.2. Z-DNA 8625 structures and/or stable RNA/DNA hybrids. Though some 3.2.3. Triplex Structures 8627 sequences that can form these unusual DNA structures are 3.2.4. Guanine Quadruplexes 8628 localized within genes (mostly in introns), the majority of them 3.2.5. R-Loops 8630 occur in genomic regions that were previously assumed to be 4. More Complex Phenomena Involving Anomalous nontranscribed.15 Thus, the recent discovery of transcription in Transcription 8631 presumed transcriptionally silent regions, such as telomeres,3 4.1. Transcription−Replication Collisions 8631 combined with the realization that most of the genome is 4.2. Transcription-Coupled Repair 8631 transcribed (reviewed in ref 16), strongly implies that these 4.3. Interdependence between Transcription sequences could be highly biologically relevant. We might and Unusual DNA Structures Mediated by expect to find many more examples in which unusual DNA Chromatin Remodeling 8632 structures would impact cellular processes as a result of their 4.4. Nascent RNA Anchoring to DNA and Its effects on transcription. Implications 8632 In this review, we discuss possible mechanisms of tran- Appendix 8633 scription through DNA sequences with unusual structural R-Loop Stability against Positive Supercoiling 8633 properties and their biological implications. We will consider Author Information 8633 primarily the elongation stage of transcription, since tran- Corresponding Author 8633 scription initiation is generally sequence-specific and often Funding 8633 involves many other proteins; thus, it is more difficult to Notes 8633 interpret the effects in terms of the physical properties of the Biographies 8633 DNA template. [Note, however, that recent data show that Acknowledgments 8634 References 8634 Special Issue: 2013 Gene Expression Received: February 6, 2013 Published: August 23, 2013 © 2013 American Chemical Society 8620 dx.doi.org/10.1021/cr400078y | Chem. Rev. 2013, 113, 8620−8637 Chemical Reviews Review alternative DNA structures could serve as promoter-like or involving nascent RNA would be expected to compete with elements.17] We will only mention briefly, in the context of this binding, thus destabilizing the transcription complex.27,28 the topic, short sequence-specific termination signals and Alternatively, this secondary structure could “push” RNA defects in the template strand, such as strand breaks and polymerase forward without RNA synthesis, thus shortening fi chemically modi ed bases. the RNA/DNA hybrid within the transcription complex instead We will begin by outlining the general possible mechanisms of disrupting the interaction between the nascent RNA and the for transcription blockage or stalling. We will then describe 29,30 exiting channel. Interestingly, although this forward various families of alternative DNA structures and their effects translocation mechanism mediated by the nascent RNA on transcription. Finally, we will consider more complex secondary structure formation has been suggested for tran- phenomena like transcription-coupled DNA repair, tran- 30 scription−replication collisions, and protein-mediated inter- scription termination at intrinsic terminators, in some cases ff actions between nascent RNA and the DNA template. nascent RNA secondary structures can have the opposite e ect: This is a new and rapidly emerging field with many models they can inhibit RNAP pausing, thus moving transcription that are still highly hypothetical. We believe that the models forward.31 It is important to note that the sequence within the and ideas suggested for one class of transcription-related events nascent RNA that forms a secondary structure interferring with might be of value for other classes. Thus, we will emphasize transcription is not necessarily completely encoded by the general mechanistic aspects of the problem, rather than DNA template; it could be partially or completely synthesized focusing upon particular biological phenomena. by reiterative transcription, in which nucleotides are repetitively added to the 3′-end of a nascent RNA due to slippage between 2. TRANSCRIPTION BLOCKAGE MECHANISMS the nascent RNA and the DNA template (e.g., refs 32−35). For Before we proceed to review transcription blockage by various example, slippage can produce quadruplex-forming oligo-G structures, it is useful to consider in general the features of sequences in nascent RNA, which interfere with further DNA or nascent RNA that could cause transcription blockage. transcription.36 During transcription, RNAP translocates along the DNA Another mechanism of interference with transcription at the template strand, synthesizing complementary RNA, and in stage of RNA extrusion could be that some extra-stable RNA/ this process, it must open up or unwind structures in which the DNA duplexes resist “peeling out” from the complex, thereby template DNA strand is involved. Thus, the most obvious 37 mechanisms would comprise obstacles for transcription temporarily immobilizing the transcription machine. localized in the template strand. They could be breaks, gaps, In contrast to DNA polymerase, RNAP normally maintains or chemically modified nucleotides, which for RNA polymerase contact with the nontemplate strand during template copying. are difficult to overcome (e.g., see refs 18, 19); alternatively, the The nontemplate strand participates in proper RNA displace- template DNA strand could be chemically intact but involved in ment and increases the processivity of transcription (see ref 27 some extrastable structure formation, which for RNA polymer- and references therein). Thus, it is possible that sequestering of ase may be difficult to unwind. These two straightforward the nontemplate strand by an unusual structure formation mechanisms, in principle, are in common for RNA polymerases could interfere with transcription.38 On the other hand, and DNA polymerases and probably for some other proteins transcription elongation can proceed in the absence of a translocating along the DNA template. complementary nontemplate strand, and at least in some However, RNA polymerase is additionally interacting with systems, the lack of the nontemplate DNA strand has only a the nontemplate DNA strand, nascent RNA, and an RNA/ very weak effect for short stretches of nucleotides with a DNA hybrid within the transcription complex; unusual 39 random sequence. Furthermore, there are no clearly defined structural properties of any of these nucleic acid moieties could affect transcription and, in particular, facilitate tran- blockages at the sites of disruptions in the nontemplate strand, also suggesting that contiguous contact with the nontemplate scription blockage. For example, an oligo-dA DNA template 40 forms an especially weak duplex with the complementary oligo- strand is not an absolute requirement for transcription. U RNA, urging RNAP to spontaneously terminate transcription However, disruptions in the nontemplate strand might strongly 40 in stretches of oligo-dT/dA, when an especially weak rU/dA exacerbate the blockages produced by other factors. Another duplex is formed within the transcription complex.20 While possible mechanism is that nontemplate-strand-mediated these stretches are very weak pausing/termination signals per interactions could alter the conformation of the neighboring se, they could be strongly exacerbated by structures formed in DNA, sterically sequestering the RNAP, thus interfering with the nascent RNA or between RNA and DNA (e.g., see refs 21, transcription.41,42 22). Interestingly, not only stability but also the shape of RNA/ Figure