Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures.
TL;DR: It is shown that the activity of BLM is substrate dependent, and highly regulated by a short ssDNA segment that separates the G4 motif from dsDNA, and a model is presented that proposes a unique role for G4 structures in modulating theActivity of DNA processing enzymes.
Abstract: Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.
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TL;DR: This Review discusses the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies, and discusses the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G 4s as therapeutic targets in human diseases.
Abstract: DNA and RNA can adopt various secondary structures. Four-stranded G-quadruplex (G4) structures form through self-recognition of guanines into stacked tetrads, and considerable biophysical and structural evidence exists for G4 formation in vitro. Computational studies and sequencing methods have revealed the prevalence of G4 sequence motifs at gene regulatory regions in various genomes, including in humans. Experiments using chemical, molecular and cell biology methods have demonstrated that G4s exist in chromatin DNA and in RNA, and have linked G4 formation with key biological processes ranging from transcription and translation to genome instability and cancer. In this Review, we first discuss the identification of G4s and evidence for their formation in cells using chemical biology, imaging and genomic technologies. We then discuss possible functions of DNA G4s and their interacting proteins, particularly in transcription, telomere biology and genome instability. Roles of RNA G4s in RNA biology, especially in translation, are also discussed. Furthermore, we consider the emerging relationships of G4s with chromatin and with RNA modifications. Finally, we discuss the connection between G4 formation and synthetic lethality in cancer cells, and recent progress towards considering G4s as therapeutic targets in human diseases.
543 citations
TL;DR: A general overview of the helicase/G-quadruplex field is presented and it is suggested that proteins may have evolved to remove these structures from genomic DNA.
Abstract: Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as 'knots' within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.
327 citations
Cites background or methods from "Mechanistic insight into the intera..."
...The mechanism employed by BLM (106,110) and Pif1 (57,163) enzymes to unfold G-quadruplex structures were investigated using smFRET techniques....
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...The mechanistic reason behind this property was elucidated with a smFRET study from the Rothenberg lab (106), in which they showed how the two RecQ-family protein domains, the RQC and the Helicase-RNase D C-terminal (HRDC) domains (107), are important for the G4 unwinding activity, and also provided a rationale to explain ATPindependent G4 unwinding....
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TL;DR: These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.
Abstract: RNA/DNA hybrids form when RNA hybridizes with its template DNA generating a three-stranded structure known as the R-loop. Knowledge of how they form and resolve, as well as their functional roles, is limited. Here, by pull-down assays followed by mass spectrometry, we identified 803 proteins that bind to RNA/DNA hybrids. Because these proteins were identified using in vitro assays, we confirmed that they bind to R-loops in vivo. They include proteins that are involved in a variety of functions, including most steps of RNA processing. The proteins are enriched for K homology (KH) and helicase domains. Among them, more than 300 proteins preferred binding to hybrids than double-stranded DNA. These proteins serve as starting points for mechanistic studies to elucidate what RNA/DNA hybrids regulate and how they are regulated.
108 citations
TL;DR: G4IPDB as discussed by the authors is the first database that provides comprehensive information about protein-Nucleic acid G-quadruplex structure (G4) interaction at a single platform.
Abstract: Nucleic acid G-quadruplex structure (G4) Interacting Proteins DataBase (G4IPDB) is an important database that contains detailed information about proteins interacting with nucleic acids that forms G-quadruplex structures. G4IPDB is the first database that provides comprehensive information about this interaction at a single platform. This database contains more than 200 entries with details of interaction such as interacting protein name and their synonyms, their UniProt-ID, source organism, target name and its sequences, ∆Tm, binding/dissociation constants, protein gene name, protein FASTA sequence, interacting residue in protein, related PDB entries, interaction ID, graphical view, PMID, author's name and techniques that were used to detect their interactions. G4IPDB also provides an efficient web-based "G-quadruplex predictor tool" that searches putative G-quadruplex forming sequences simultaneously in both sense and anti-sense strands of the query nucleotide sequence and provides the predicted G score. Studying the interaction between proteins and nucleic acids forming G-quadruplex structures could be of therapeutic significance for various diseases including cancer and neurological disease, therefore, having detail information about their interactions on a single platform would be helpful for the discovery and development of novel therapeutics. G4IPDB can be routinely updated (twice in year) and freely available on http://bsbe.iiti.ac.in/bsbe/ipdb/index.php.
82 citations
TL;DR: It is reported that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession.
Abstract: G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.
80 citations
Cites background or result from "Mechanistic insight into the intera..."
...The BLM binding and GQ-unfolding activity was independent of ATP, similar to the case of RHAU, which is in agreement with previous studies (9, 27, 28)....
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...Recent single-molecule studies have reported that truncated BLM interacts with telomeric GQ (9, 27, 28)....
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...BLM exhibited a similar FRET fluctuation, in agreement with previous studies (28, 29), which indicates that a similar mechanism is at work (Fig....
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...Although the ATP-independent unfolding activity by RHAU and BLM that we present here is consistent with some previous studies that reported ATP independent GQ unwinding activity by BLM (9, 28), others have presented ATPdependent unwinding by RHAU and BLM....
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References
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TL;DR: It is speculated that this self-recognition of guanine-rich motifs of DNA serves to bring together, and to zipper up in register, the four homologous chromatids during meiosis.
Abstract: We have discovered that single-stranded DNA containing short guanine-rich motifs will self-associate at physiological salt concentrations to make four-stranded structures in which the strands run in parallel fashion. We believe these complexes are held together by guanines bonded to each other by Hoogsteen pairing. Such guanine-rich sequences occur in immunoglobulin switch regions, in gene promoters, and in chromosomal telomeres. We speculate that this self-recognition of guanine-rich motifs of DNA serves to bring together, and to zipper up in register, the four homologous chromatids during meiosis.
1,562 citations
TL;DR: There is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.
Abstract: Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.
1,493 citations
TL;DR: From examination of the optical properties of the gel and investigation of the structure of fibers obtained from the gel by drying, it is concluded that, at least in the case of the 5' isomer, the phenomenon may be explained as being due to helix formation by the guanylic acid.
Abstract: In 1910, Bang' reported that concentrated solutions of guanylic acid formed a gel. We have also observed that concentrated solutions (25.0 mg/ml) of guanylic acid (GMP) at pH 5 are extremely viscous and, if cooled, form a clear gel. Less concentrated solutions also gel on cooling but assume a more normal viscosity at room temperature. From examination of the optical properties of the gel and investigation of the structure of fibers obtained from the gel by drying, we have concluded that, at least in the case of the 5' isomer, the phenomenon may be explained as being due to helix formation by the guanylic acid. A possible structure is presented for this helix.
1,327 citations
TL;DR: This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.
Abstract: In addition to the canonical double helix, DNA can fold into various other inter- and intramolecular secondary structures. Although many such structures were long thought to be in vitro artefacts, bioinformatics demonstrates that DNA sequences capable of forming these structures are conserved throughout evolution, suggesting the existence of non-B-form DNA in vivo. In addition, genes whose products promote formation or resolution of these structures are found in diverse organisms, and a growing body of work suggests that the resolution of DNA secondary structures is critical for genome integrity. This Review focuses on emerging evidence relating to the characteristics of G-quadruplex structures and the possible influence of such structures on genomic stability and cellular processes, such as transcription.
1,176 citations
TL;DR: It is proposed that the G-quartet structure must be dealt with in vivo by the telomere replication machinery, hydrogen-bonded structures formed from four guanosine residues in a square-planar array.
1,096 citations