G-Quadruplex Unwinding Helicases and Their Function in Vivo

Biochemical Society Transactions (2017) DOI: 10.1042/BST20170097

Review Article G-quadruplex unwinding helicases and their function in vivo

Markus Sauer and Katrin Paeschke European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands Correspondence: Katrin Paeschke ([email protected])

The concept that G-quadruplex (G4) structures can form within DNA or RNA in vitro has been long known and extensively discussed. In recent years, accumulating evidences imply that G-quadruplex structures form in vivo. Initially, inefficient regulation of G-quadruplex structures was mainly associated with genome instability. However, due to the location of G-quadruplex motifs and their evolutionary conservation, different cellular functions of these structures have been postulated (e.g. in telomere maintenance, DNA replication, transcription, and translation). Regardless of their function, efficient and controlled formation and unwinding are very important, because ‘mis’-regulated G-quadruplex structures are detrimental for a given process, causing genome instability and diseases. Several helicases have been shown to target and regulate specificG-quadruplexstructures.This mini-review focuses on the biological consequences of G4 disruption by different helicases in vivo.

Introduction G-quadruplex (G4) structures can form in DNA and RNA molecules when harboring a specific con- sensus motif (G≥3N1-7G≥3N1-7G≥3N1-7G≥3). Recently, different in vitro and in vivo experiments show that variations in this motifs are also possible underlining the diversity of this structure (e.g. with G2 tracks or divided by a variable number of nucleotides) [1]. Four guanines are cyclically coordinated by Hoogsteen base pairing in a planar arrangement termed as G-quartet [2]. Stacking of these G-quartets results in a conformational diverse three-dimensional structure called G4 [3]. In silico analyses identified G4 motifs in various organisms and revealed that they are not randomly distributed in the genome. Instead, they are overrepresented in areas of regulatory relevance like transcriptional start sites and promoters, meiotic and mitotic double-strand break sites, replication hot- spots, and telomeres (reviewed in refs [4,5]). Additionally, G4 motifs and their genomic locations are evolutionarily conserved [6,7], suggesting that G4 structures potentially influence biological processes (reviewed in ref. [8,9]). On the RNA level, G4 motifs are enriched in the 50- and 30-UTRs of mRNAs as well as in many noncoding transcripts [10]. RNA-G4 structures perform functions in processes like miRNA biogenesis, telomere maintenance, and translation (reviewed in refs [11,12]). Although the existence and functions of G4 structures in vivo are controversially discussed, numerous experiments such as antibody staining (Sty49, BG4, and D1) [13–15] and genetic and genomewide analyses show that G4 structures do form in vivo and bear diverse biological functions (reviewed in ref. [16]). Different studies in vitro and in vivo have shown that misregulation of G4 structures (uncontrolled formation or disruption) leads to changes in cellular processes (reviewed in refs [17,18]), resulting in genome instability and diseases. G4 structures can act as roadblocks to active rep- Received: 30 June 2017 lication as well as to the transcriptional and translational machinery, and hence need to be unwound Revised: 31 July 2017 to maintain genome and cellular output. Various G4-interacting proteins have been identified in Accepted: 10 August 2017 numerous studies (reviewed in ref. [19]). Among the identified proteins were multiple helicases that Version of Record published: specifically unwind G4 structures in vitro. To date, 15 eukaryotic helicases have been described to 22 September 2017 unwind G4 structures, differing in strand and target specificity (Table 1). They target G4 structures

during different biological processes and hence affect genome instability on many levels. For a detailed review on the mechanisms of helicases, see ref. [20]. Here, we will discuss the helicases which are able to unwind G4 structures in vivo and what the biological consequences of this regulation are.

Effects of G4 helicases during telomere maintenance G4 structures form within telomeric DNA, telomerase RNA component (TERC), internal telomeric repeats, and telomeric repeat-containing RNA (TERRA). Owing to these different locations and conformational diversity [1,21,22], G4 structures are able to influence telomere maintenance in multiple ways: protection from deg- radation, support of nuclear organization, and alteration of telomerase activity (reviewed in refs [9,16]). Consequently, many helicases have been implicated to affect G4 unfolding at telomeres (Figure 1). RecQ helicases are 30–50 DNA helicases present in numerous organisms. Deletion or mutation of the human RecQ family members is associated with genetic disorders and cancer predisposition (reviewed in ref. [23]). It has been demonstrated in vitro that most RecQ helicases can unwind G4 structures, although with different specificity and processivity [24–27]. The human RecQ helicase family members WRN and BLM support telomere replication in vivo, which is speculated to be related to G4 structure unwinding [24,28]. Without WRN, defects during lagging-strand replication and end-to-end fusions of telomeres are observed [29]. BLM, which has greater G4 unwinding activity than WRN, supports telomeric replication on the leading strand. In vivo, loss of BLM activity results in a significant enrichment of G4 structures, especially at telomeres, and altered replication speed. This slow-down of replication is further enhanced upon the addition of the G4-stabilizing ligand Phen-DC3 [30]. Interestingly, in BLM-deficient cells, such as in the disease Bloom syndrome, fragile telomeres are observed at sister chromatids derived from G-rich templates [31–33]. In ciliates, a RecQ-like helicase was shown to regulate G4 structures during DNA replication together with telomerase. This results in detach- ment of the telomeres from the nuclear scaffold and permits telomere elongation [34–36]. RTEL1 is another helicase, which is connected to G4 structure unwinding at telomeres. This 50–30 DNA helicase, identified in worm, mouse, and mammals, belongs to the Rad3/XPD family and is described to be essential for telomere maintenance and DNA repair [37,38]. In the absence of RTEL1, telomeres are short [38] and become fragile [31] causing the rare genetic disorder Hoyeraal-Hreidarsson syndrome [39]. This telomeric defect is even more severe upon the addition of the G4 ligand TMPyP4 [38,40]. Interestingly, no dramatic effect on telomeres is known for other family members (XPD, FANCJ, Dog-1, or ChlR1; [41]). ATRX is an SWI2/SNF2 DNA helicase/ATPase [42], which supports replication fork progression, influences telomere length, and targets G4 structures [43,44]. This led to the speculation whether these three observations are linked to each other. A recent study showed that ATRX targets R-loops in transcribed telomeric repeats. The authors suggest that ATRX averts G4 formation within these R-loops and by this prevents replication stal- ling and DNA damage at telomeres [44]. Nonetheless, additional studies in vivo are required to fully address this statement. Another DNA helicase, which is implicated in telomere biology and DNA replication (flap maturation during Okazaki fragment maturation), is Dna2. In vitro experiments showed that Dna2 can recognize and disrupt telomeric G4 structures [45]. In Dna2-deficient mouse cells, fragile telomeres and severe telomere replication problems occur. This telomere phenotype is increased upon the addition of TMPyP4 or TMS, both G4 stabilization components [45]. The 30–50 RNA helicase DHX36 (RHAU or G4R1) belongs to the DEAH box family and was identified as the major source for G4 unwinding activity in HeLa cell lysate [46]. In vitro analyses confirmed resolving of DNA- and RNA-G4 structures by DHX36 [47,48]. The formation of a G4 at the 50-end disrupts an essential structure within TERC (the P1 helix) and alters telomerase activity [49,50]. DHX36 binds to this G4 structure in vitro [51]. In DHX36-knockdown HEK293T cells, telomerase activity is down-regulated and telomeres are not elongated [52,53]. This finding led to the model that DHX36 regulates telomerase function/processivity by unwinding the G4 structure within TERC. Different studies in vivo have shown that TERRA, the product of transcription at telomeres, has a major impact on telomere length regulation and telomerase function (reviewed in ref. [54]). TERRA was shown to fold into G4 structures in vitro [55,56]. So far, no helicase involved in TERRA-G4 regulation has been described. A likely candidate is DHX36, because of its presence at telomeres and its specificity to unwind RNA-G4 structures. To date, it is clear that telomere biology strongly depends on the function of helicases and that helicases, especially in the context of G4s, support telomere maintenance at multiple levels.

Table 1 Helicases implicated to regulate G4 in vivo List of all helicases and selected features, which have been described to regulate G4 structure unwinding in vivo. Telomere DNA Name Family Processivity Specificity Organism maintenance replication Transcription Translation Genetic diseases

Pfh1 Pif1-like 50–30 DNA S. pombe ?Yes Pif1 Pif1-like 50–30 DNA S. cerevisiae ?Yes DHX36 DEAH 30–50 DNA, RNA Homo sapiens Yes Yes Yes WRN RecQ-like 30–50 DNA H. sapiens Yes Yes Yes Werner syndrome BLM RecQ-like 30–50 DNA H. sapiens Yes Yes Yes Bloom syndrome Sgs1 RecQ-like 30–50 DNA S. cerevisiae ? Only in vitro Yes FANCJ Rad3/XPD 50–30 DNA H. sapiens Yes Fanconi anemia Dog-1 Rad3/XPD 50–30 DNA C. elegans Yes RTEL1 DEAH 50–30 DNA H. sapiens Yes Yes Hoyeraal-Hreidarsson syndrome ATRX Swi2/Snf2 50–30 DNA H. sapiens Yes ATRX syndrome Dna2 Upf1-like 50–30 DNA S. cerevisiae Yes ? DDX21 DEAD 50–30 DNA, RNA H. sapiens Yes DHX9 DEAH 30–50 DNA, RNA H. sapiens Yes? Yes? XPD Rad3/XPD DNA H. sapiens Yes Xeroderma (XPB) pigmentosum eIF4A DEAD RNA H. sapiens Yes Cancer

Effects of helicases operating on G4 structures during replication G4 structures can form not only at telomeres but also at internal genomic regions [57–59]. It has been shown in human cells that G4 structure formation is stimulated during S-phase [4,14,60]. Different models have been postulated for the biological function of G4 structures during DNA replication. At origins, G4 structures have been shown to support the initiation of DNA replication (reviewed in ref. [61]). Alternatively, G4 structures might safeguard proper replication or prevent uncoupling of the leading- and lagging-strand polymerases. Contrary, G4 structures are an obstacle for the replication fork machinery and are in need of unwinding. In agreement with this is the fact that G4 structures cause genomic and epigenetic instability if they are not efficiently regulated [62,63]. So far, a few helicases have been discussed in the context of DNA replication and genome stability (Figure 1). The first helicase that has been shown to regulate G4 structures in vivo was the 50–30 DNA helicase DOG-1 of the Rad3/XPD family. Well-designed studies in Caenorhabditis elegans showed that DOG-1 is essential for genome stability at G4 motifs. Upon mutation of DOG-1, genetic alterations and deletions accumulated within these G4 motifs [64,65]. The closely related helicase FANCJ (BRIP1 or BACH1) was shown to support replication through G4 structures, as demonstrated in Xenopus egg extracts [66]. Note that mutations of FANCJ are connected to the human chromosomal instability disorder Fanconi anemia, but this is most likely not directly linked to its role in regulating G4 structures [67]. Detailed studies in vitro and in vivo revealed that FANCJ supports DNA replication [68] and can unwind G4 structures in vitro [69,70]. FANCJ promotes genome stability at G4s by two independent mechanisms: FANCJ unwinds G4 structures that have been destabilized by the translesion synthesis polymerase REV1. Second, WRN or BLM assists FANCJ in unwinding G4 structures from both directions [71–73]. Interestingly, in the absence of FANCJ, genomic deletions at G4 motifs were identified [69]. Pif1 helicase family members have been shown to efficiently unwind G4 structures in vitro with a higher specificity than other helicases [74–78]. These 50–30 DNA helicases are conserved from yeasts to human [79]. Genome-wide and molecular studies have shown that ScPif1 (Saccharomyces cerevisiae) and Pfh1 (Saccharomyces pombe) bind to G4 motifs and specifically support local DNA replication at these regions [76,80]. Two different genetic assays in yeast, one using a G-rich tandem array consisting of human minisatellites (CEB1) and one using inserted G4 motifs, revealed that in the absence of ScPif1, G4 motifs are unstable

Figure 1. Involvement of G-quadruplex helicases in cellular processes in vivo. Cellular processes discussed in the present paper are indicated with their cellular localization (nuclear = yellow background, cytosolic = red background). G4 structure-resolving helicases are listed according to their contribution to each process. Locations of potential G4-forming events are listed in corresponding bubbles. Top section = telomere maintenance (yellowish proteins): G4s can form in telomeric DNA, TERRA, and TERC. Middle section = DNA replication (reddish proteins): G4s can form in the leading strand, lagging strand, and at origins. Bottom left section = transcription (RNA pol II in blue, gray, and turquoise represents introns, respectively, exons of mRNAs): G4s can form in TSSs, promoters, and at the 30-end of genes. Bottom right section = translation (ribosomes in green, turquoise in mature mRNA, and translated proteins in dark gray): G4s can form in the 50-UTR, 30-UTR, and the coding sequence of mRNAs.

and gross chromosomal rearrangement increases significantly [74,78,80–82]. Pif1 binds and unwinds G4 structures at the end of S-phase [80]. To date, it is not clear if ScPif1 functions on G4 structures on both replicating strands or has preferential strand specificity. Studies using CEB1 have shown that ScPif1 supports genome stability only at G4 structures located on the leading strand [81], whereas other studies revealed that ScPif1 binds and regulates G4 structures on both strands [74,80]. These controversial data can be explained that under different experimental conditions, diverse and different G4 structure conformations occurred and that other factors in addition to ScPif1 were needed to aid G4 regulation. This speculation agrees with a recent study in yeast, showing that Mms1, a ubiquitin ligase partner, supports ScPif1 binding to G4 structures located on the lagging strand [83]. The previously mentioned RecQ helicase family is also known to influence multiple steps during DNA replication, to interact with multiple DNA repair proteins, and to unwind G4 structures efficiently (reviewed in ref. [84]). Nevertheless, direct evidence that RecQ helicases regulate G4 structure formation during DNA replication in vivo is missing. Many experiments have revealed that replication fork progression is negatively affected at G4 motifs and that this is enhanced in the absence of helicases. It remains elusive why G4 structures form during DNA replication. As mentioned above, replication initiation or control of replication fork progression might be positive scenarios,

but no helicase is linked to G4 structures formed within origins of replication, yet. Potential candidates might be RECQ1 and RECQ4, which are associated with origin function [85]. Effects of helicases acting on G4 structures during transcription G4 motifs are enriched at transcriptional start sites (TSSs) and promoter regions of genes thereby providing a decisive mechanism for transcription initiation [86]. Of great interest is the fact that especially in promoter regions of onco- and regulatory genes, G4 motifs are enriched, whereas in tumor suppressor and housekeeping genes they are depleted [86,87]. This fact provides a logical connection between G4-dependent gene regulation and carcinogenesis. G4 structures are postulated to affect transcription on multiple levels: they reduce transcription by blocking polymerases, stimulate transcription by recruiting proteins, and keep the nascent strand in a single-stranded conformation by G4 structure formation on the non-transcribed strand [9,88]. In agreement with this are in cellulo and in vivo studies showing transcriptional changes upon formation of G4 structures by chemical ligands [89–92]. So far, the best described G4 structure-mediated effect on transcription comes from reports studying the promoter region of the proto-oncogene c-MYC [93]. Changes in c-MYC transcription levels are associated with G4 formation as observed by in vivo footprinting and other molecular analyses [58,94–98]. Although there is a lot known about G4 structure formation and its impact on c-MYC transcription, only little is known about the ‘disruption’ of these G4 structures. So far, only three proteins, among them one helicase, have been described to support unfolding of G4 structures in c-MYC: CNBP, PARP-1, and Pif1 [99–101]. Nevertheless, a detailed analysis of these proteins and changes in G4-mediated c-MYC transcription are missing. RecQ helicase family members are also assumed to regulate transcription by resolving DNA-G4 structures. The S. cerevisiae RecQ helicase Sgs1 and the above-introduced human BLM and WRN helicases can unwind G4 structures [102,103]. Gene expression changes in cells lacking these helicases are observed specifically at regions that harbor a G4 within the promoter region/TSS [90,104,105]. Two members of the Rad3/XPD family, XPD and XPB, are also proposed to affect transcription via G4 structure unfolding. XPB binds to G4 structures, whereas XPD binds and unwinds G4 structures in vitro. Interestingly, genome-wide ChIP-seq analyses of these two proteins in human cells revealed that 40% of their binding sites overlap with G4 motifs. XPD and XPB bind particularly to G4 structures at promoters of highly transcribed genes. The authors speculate that XPD and/or XPB support transcription of genes linked to diseases by unwinding local G4 structures [106]. Another helicase that was described to have a function at G4s in promoter regions is the RNA helicase DHX36. YY1 harbors G4 motifs in its promoter and in the 50-UTR of its mRNA. The YY1 gene encodes for a multifunctional protein with regulatory potential in tumorigenesis [107]. At both loci, G4 structures affect YY1 expression. Surprisingly, DHX36 acts only on DNA-G4 structures located in the promoter but not on putative RNA-G4 structures in the 50-UTR [108]. G4 motifs are overrepresented immediately after the 30-end of genes. This leads to a model in which G4 structures assist the correct termination of transcription. To this end, G4 structures are pausing sites for the polymerases, which ultimately result in the termination of transcription [109,110]. In this model, G4 structures can be formed by the RNA transcript [111] or by a DNA–RNA hybrid [112–114]. A good candidate for regulating this mechanism is the DEAH box helicase DHX9. DHX9 can resolve RNA-G4s nearly twice as fast as their DNA analog in vitro [115]. Furthermore, it was described as an abundant and RNA polymerase-associated protein. However, regulation of transcription by DHX9 in vivo was only demonstrated in context of other mechanisms, mainly by resolving RNA forks and R-loops resulting in fastened polymerase progression (reviewed in ref. [116]). As it becomes more obvious that G4 structures potentially influence transcription positively and negatively, it is less clear how they form or are unfolded and what are the biological consequences of G4 structure formation and regulation. G4 regulation and the consequences for RNA-mediated processes Lacking a competing complementary strand, G-rich RNA is even more prone to fold G4 structures than DNA. Additionally, G4 structures have an even higher thermodynamic stability in RNA [117]. During post- transcriptional gene regulation, G4 structures can be present within the 50-or30-UTR or the coding region of mRNAs. This results in numerous potential roles for G4 during protein synthesis (reviewed in ref. [12]). Although many different RNA-G4-interacting proteins have been identified [118], only a few RNA helicases are known to regulate G4 structures. A recent study in HEK293 cells came to the conclusion that in these cells,

G4 structures are not present at the mRNA level, because they are rapidly removed by an ATP-independent mechanism [119]. Yet, there are numerous studies relating a molecular function of mRNAs with G4 structures, which will be presented below (Figure 1). DHX36 was shown to support translation of specific loci by unwinding G4 structures. In complex with Aven, DHX36 aids translation of the mRNAs MLL1 and MLL4 by binding to regions harboring G4 motifs [120]. Furthermore, experiments in mouse heart tissue showed that DHX36 knockout causes severe defects in cardiogenesis. Here, an unresolved G4 structure in the 50-UTR of the NKx2-5 mRNA prevents its translation and by this the expression of this important early transcription factor [121]. DHX36 also impedes the translation of the PITX mRNA, which exhibits G4 motifs in its 30-UTR. However, this seems not to be a consequence of unwinding by DHX36 but a miRNA-mediated mechanism [122]. RNA helicases regulate gene expression not only directly during translation but also by other processes which then downstream influence protein synthesis. Upon UV-induced DNA damage, DHX36 is necessary to maintain p53 pre-mRNA 30-end processing by regulating a G4 structure within the p53 mRNA. Depletion of DHX36 results in loss of p53 protein [123]. Furthermore, DHX36 was described to be involved in mRNA turn- over and localizes to stress granules [124]. Here, it is possible that DHX36 assists in the decay of G4 motif- harboring mRNAs and prevents them from being translated. The closely related helicase DHX9 was shown to unwind G4 structures in vitro [115]. DHX9 is located in both the nucleus and the cytoplasm, and was previously described to facilitate translation of mRNAs with highly structured 50-UTRs [116]. The direct link between G4 structures, DHX9, and translation has not yet been demonstrated in vivo. DDX21 is a DEAD-box RNA helicase that was recently shown to unwind G4 structures. DDX21 acts on RNA-G4 within the 30-UTR of mRNAs, and its depletion abolishes protein translation from its target mRNAs [125]. The helicase eIF4A also belongs to the DEAD-box helicase family and is another important factor imple- mented in translational processes. It was reported that RNA-G4s cause eIF4A-dependent translation of onco- genes [126]. However, whether this helicase can directly resolve G4 structures has not been shown so far. The biology of RNA-G4 structures is less studied than its DNA counterpart. However, it is obvious that RNA-G4s have a strong impact on various steps during RNA-mediated processes (e.g. translational initiation, RNA stability, localization, and transport). Over 70 RNA helicases have been described (according to the http://www. rnahelicase.org/namingcode.htm database), but only a few have been tested on G4 substrates in vitro and in vivo. To address the question, which helicases act on RNA-G4 structures and when, is the subject of current research. Conclusion In recent years, it became clear that G4 structure formation is more complex than original anticipated. Whereas some years ago only conventional G4 motifs were discussed to fold into G4s, now less stringent motifs are shown to fold into these structures. Additionally, the topology of G4 structures themselves is highly diverse and, for the same sequence, multiple G4 conformations are possible. This diversity raises multiple open questions: have we identified all G4 unwinding helicases? How do helicases recognize different G4 structures? What is the biological mechanism how G4 structures are disrupted in vivo? Are assessor proteins involved in G4 targeting? How much impact have, in addition to helicases, other proteins in G4 disruption? If we assume that there are over 700 000 potential G4 structures in the human genome, it is unlikely that they form at once, therefore a highly interesting question is when and why and which G4 structures form in a given biological process.

Abbreviations G4, G-quadruplex; TERC, telomerase RNA component; TSSs, transcriptional start sites.

Funding Work in the Paeschke Laboratory is funded by the European Research Council Stg Grant [638988-G4DSB] and an Emmy Noether Fellowship (Deutsche Forschungsgemeinschaft).

Acknowledgements We thank Stefan Juranek and Satyaprakash Pandey for careful reading of the manuscript.

Competing Interests The Authors declare that there are no competing interests associated with the manuscript.

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