G-quadruplex In Presence Research Articles

Transactivation by partial function P53 family mutants is increased by the presence of G-quadruplexes at a promoter site

The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

Benzoselenazolium-based hemicyanine dye for G-Quadruplex detection

Benzothiazolium and benzoxazolium are common groups for the construction of hemicyanine dyes; however, their isosteric analogue benzoselenazolium have rarely been studied. Here, we report the development of the first benzoselenazolium-based hemicyanine dye for the selective detection of G-quadruplexes. This molecule, SEMA-1, was validated as a red-emitting and activatable fluorescent probe whose fluorescence would only be activated in the presence of G-quadruplexes in buffer solution. Consistent with this, SEMA-1 was found to accumulate in nucleoli and could be used to detect the high abundance of nucleolar rDNA and rRNA G-quadruplexes in fixed HeLa cells. On the other hand, due to the retained mitochondrial membrane potential in live HeLa cells, SEMA-1 was captured by mitochondria and had the potential to detect the mitochondrial G-quadruplexes. Collectively, this work demonstrates the value of developing G-quadruplex-specific fluorescent probes from novel benzoselenazolium-based hemicyanine scaffold.

Unified-amplifier based primer exchange reaction (UniAmPER) enabled detection of SARS-CoV-2 from clinical samples

Primer exchange reaction (PER) is an emergent method for non-templated synthesis of single stranded DNA molecules. PER has been shown to be effective in cell imaging systems and for detection of macromolecules. A particular application of PER is to detect a specific target nucleic acid. To this endeavor, two coupled DNA hairpins, a detector and an amplifier, play in accordance to extend a target nucleic acid with a concatemer DNA sequence. Here we introduced unified-amplifier based primer exchange reaction (UniAmPER) that beneficially extends the target by a unified-amplifier. The unified-amplifier operates as both detector and amplifier hairpins. The extension resulted in synthesis of concatemer G-rich sequences. The G-rich sequences were expected to form G-quadruplex (GQ) structures. Presence of the GQ structures were investigated by peroxidase activity of GQs in presence of hemin, H2°2 and 3,3′,5,5′-Tetramethylbenzidine (TMB) as well as by fluorescence signal generation upon intercalation of thioflavin T (ThT). The presented unified-amplifier in this study facilitates application of PER systems for development of colorimetric or fluorogenic biosensors. As a proof of principle, the method has been applied for detection of reversely transcribed cDNAs from clinical SARS-CoV-2 samples.

Encounters between Cas9/dCas9 and G-Quadruplexes: Implications for Transcription Regulation and Cas9-Mediated DNA Cleavage.

Using the nuclease-dead Cas9 (dCas9), we targeted in cellulo a G-rich sequence, which contains multiple potentially G-quadruplex (GQ) forming sites, within the human tyrosine hydroxylase (TH) promoter. We demonstrate that transcription can be up or down regulated by targeting different parts of this G-rich sequence. Our results suggest that TH transcription levels correlate with stability of different GQs formed by this sequence and targeting them with dCas9 can modulate their stability. Unlike alternative approaches, regulating TH expression by targeting the promoter GQs with dCas9 enables a specific and potentially transient control and does not require mutations in the sequence. We also investigated whether the presence of GQs in target sequences impacts DNA cleavage activity of Cas9. We discovered significant reduction in cleavage activity when the vicinity of a high-stability GQ was targeted. Furthermore, this reduction is significantly more prominent for the G-rich strand compared to the complementary C-rich strand.

Biological relevance and therapeutic potential of G-quadruplex structures in the human noncoding transcriptome.

Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

Investigating C-kit1 G-Quadruplex Stability using Nanopore

In an aqueous solution, certain sequences of single-stranded DNAs can fold into dynamic secondary structures, whose stabilities vary with their surrounding environment contents. In this study, we investigated the C-kit1 promoter - a sequence that plays an important role in several human malignancies. C-kit1 is a guanine-rich sequence that can form stable G-quadruplex structure in the presence of cations. In the presence of G-quadruplex on the promoter region, C-kit1 gene is downregulated, leading to suppression of cancer cell proliferation. Our study aimed to evaluate C-kit1 G-quadruplex structural stability dependence on cations and CX-5461, a small G-quadruplex stabilizing agent that is currently in Phase 1 clinical trial. Conventional methods often utilize fluorescence spectroscopy and circular dichroism to study CX-5461 efficiency on stabilizing G-quadruplex. However, these methods cannot provide real-time molecular dynamic sensing of the structure folding/unfolding behavior. Thus, our study utilized the α-hemolysin nanopore as the main tool for single-molecular analysis of C-kit1 G-quadruplex. Specifically, C-kit1 G-quadruplex formation and stability with and without CX-5461 presence was analyzed using α-hemolysin nanopore, circular dichroism and thermal denaturation. Circular dichroism spectra measurements contained a positive peak at 263nm and negative peak at 240nm, confirming G-quadruplex formation with anti-parallel topology in both 1M KCl and 1M NaCl. Nanopore analysis results showed that C-kit1 G-quadruplex stability is cation-dependent (K+>Na+), resulting in in longer current-blockage events in K+ and significantly decreased event capture rate. Addition of CX-5461 drug further increased C-kit1 G-quadruplex stability, resulting in extended current-blockage events (seconds to minutes long) in nanopore study, and a significantly increased melting temperature.

Methods for Identification and Validation of G-Quadruplex Sequences in Legumes.

Among the different secondary structures that DNA can adopt, G-quadruplex is a noncanonical form that has recently started to garner attention about the possible layers of regulation they could introduce in cellular processes. Here, we outline how the presence of G-quadruplexes can be probed in legumes and other plant genomes. This chapter describes various in silico approaches that can be utilized to identify putative G-quadruplex forming sequences (GQSes) and validate their formation through in vitro experimental approaches.

A secondary structure within a human piRNA modulates its functionality

The piwi-interacting RNAs (piRNAs) are small non-coding RNAs, mostly 24–32 nucleotides in length. The piRNAs are not known to have any conserved secondary structure or sequence motifs. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ) forming sequences in human piRNAs. We studied human piR-48164/piR-GQ containing a potential GQ forming sequence and using biochemical and biophysical techniques confirmed its ability to form a GQ. Using EMSA, we discovered that the formation of GQ structure led to inhibition of the piRNA binding to the HIWI-PAZ domain as well as the complementary base pairing to a target RNA. The inability of the piR-GQ to interact with the PIWI protein might be detrimental to the function of the piRNA. To investigate if the formation of a GQ structure in piRNA prevents its target gene silencing in vivo, we used a reporter assay. The piR-GQ failed to inhibit the reporter gene expression while a mutated version that lacked the ability to form GQ inhibited reporter gene expression indicating that the presence of GQ in piRNA is detrimental to its function. These studies unraveled the dependence of a piRNA's functionality on an RNA secondary structure and added a new layer of regulation to their function.

G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing

Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5′ region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing.

Probing the Potential Role of Non-B DNA Structures at Yeast Meiosis-Specific DNA Double-Strand Breaks

A plethora of evidence suggests that different types of DNA quadruplexes are widely present in the genome of all organisms. The existence of a growing number of proteins that selectively bind and/or process these structures underscores their biological relevance. Moreover, G-quadruplex DNA has been implicated in the alignment of four sister chromatids by forming parallel guanine quadruplexes during meiosis; however, the underlying mechanism is not well defined. Here we show that a G/C-rich motif associated with a meiosis-specific DNA double-strand break (DSB) in Saccharomyces cerevisiae folds into G-quadruplex, and the C-rich sequence complementary to the G-rich sequence forms an i-motif. The presence of G-quadruplex or i-motif structures upstream of the green fluorescent protein-coding sequence markedly reduces the levels of gfp mRNA expression in S. cerevisiae cells, with a concomitant decrease in green fluorescent protein abundance, and blocks primer extension by DNA polymerase, thereby demonstrating the functional significance of these structures. Surprisingly, although S. cerevisiae Hop1, a component of synaptonemal complex axial/lateral elements, exhibits strong affinity to G-quadruplex DNA, it displays a much weaker affinity for the i-motif structure. However, the Hop1 C-terminal but not the N-terminal domain possesses strong i-motif binding activity, implying that the C-terminal domain has a distinct substrate specificity. Additionally, we found that Hop1 promotes intermolecular pairing between G/C-rich DNA segments associated with a meiosis-specific DSB site. Our results support the idea that the G/C-rich motifs associated with meiosis-specific DSBs fold into intramolecular G-quadruplex and i-motif structures, both in vitro and in vivo, thus revealing an important link between non-B form DNA structures and Hop1 in meiotic chromosome synapsis and recombination.

Quantum Dots-Ligand Complex as Ratiometric Fluorescent Nanoprobe for Visual and Specific Detection of G-Quadruplex.

By complexing a nonionic G-quadruplex ligand with hybrid dual-emission quantum dots (QDs), a ratiometric fluorescent nanoprobe is developed for G-quadruplex detection in a sensitive and specific manner. The QDs nanohybrid comprised of a green-emission QD (gQD) and multiple red-emission QDs (rQDs) inside and outside of a silica shell, respectively, is utilized as the signal displaying unit. Only the presence of G-quadruplex can displace the ligand from QDs, breaking up the QDs-ligand complexation, and inducing the restoration of the rQDs fluorescence. Since the fluorescence of embedded gQD stays constant, variations of the dual-emission intensity ratios display continuous color changes from green to bright orange, which can be clearly observed by the naked eye. Furthermore, by utilizing competitive binding of a cationic ligand versus the nonionic ligand toward G-quadruplex, the nanoprobe is demonstrated to be applicable for assessing the affinity of a G-quadruplex-targeted anticancer drug candidate, exhibiting ratiometric fluorescence signals (reverse of that for G-quadruplex detection). By making use of the specificity of the ligand binding with G-quadruplex against a double helix, this nanoprobe is also demonstrated to be capable of sensitive detection of one-base mutation, exhibiting sequence-specific ratiometric fluorescence signals. By functionalizing with a nuclear localization peptide, the nanoprobe can be used for visualization of G-quadruplex in the nucleus of human cells.

Abstract 5418: Lipid modification of c-MYC promoter targeted oligonucleotide stabilizes G-quadruplex formation and enhances its growth inhibitory activity in leukemia cells

Abstract Deregulation c-MYC is considered to be the hallmark of cancer as it regulates a large array of key genes essential for cell function such as proliferation, metabolism, differentiation, adhesion and apoptosis. Consequently, c-MYC expression is tightly regulated at the transcriptional and post-transcriptional levels. One of the key regulators of c-MYC transcription is the Nuclease Hypersensitive Element III1 (NHEIII1) located up-stream of P1 promoter. NHEIII1 is a gene silencer requiring for its function a stable spatial conformation that is provided by the high G/C rich ratio that forms G-quadruplex and i-motif structures. The oligonucleotide sequence Pu27 (27 nucleotides) forms G-quadruplex and correspond to the genomic NHEIII1 sequence. We have shown that Pu27 reduces c-MYC transcription in leukemia cell lines and consequently inhibits cell growth and promotes apoptosis. In this study, we evaluated the effect of Pu27 modification using polyethylene glycol (PEG), tocopherol (Toco) and the lipid palmitate (Palmi) in order to increase G-quadruplex stability and lessen blood clearance. We investigated the binding property to NHEIII1 target sequence using electrophoretic mobility shift assay (EMSA). The target used for this study was a 134bp covering NHEIII1 as double stranded (ds) and C-rich or G-rich single stranded (ss) DNA. We verify the presence of G-quadruplex formation in the modified Pu27 using circular dichroism (CD) spectrometry in two different salinity conditions 100mM KCl: that favors G-quadruplex formation and 100mM NaCl that does not. In addition, the effect of the modified-Pu on cell proliferation was investigated in U937 and Raji leukemia cell lines using MTT assay. We show that Pu27 binds specifically to the target sequence on the C-rich strand. The CD spectra confirm the presence of G-quadruplex in the modified Pu27. Furthermore, the CD measurement in two different salinity conditions revealed that Toco and Palmi- modifications stabilize the formation of G-quadruplex even in less favorable condition (100mM NaCl). The dose response study in MTT assay reveals that Toco and Palmi modified-Pu inhibit cell growth for Raji and U937 leukemia cell lines as well, if not better than unmodified Pu27. This data demonstrates the specific binding of the G-quadruplex lipid-modified Pu27 at the silencer element in a sequence specific manner (by strand invasion) and down regulation of c-MYC expression as is the case for unmodified Pu27. Subsequently down regulation of c-MYC by Pu27 was followed by change in c-MYC target genes expression and inhibition of cell growth. Our finding suggests that modification of the c-MYC targeted oligonucleotide by addition of lipids stabilizes the 3D structure (G-quadruplex) and improve its function at inhibiting cell growth most likely by down-regulating c-MYC. Citation Format: Francine Francine, Shelia D. Thomas, Gilles Tapolsky, Donald M. Miller. Lipid modification of c-MYC promoter targeted oligonucleotide stabilizes G-quadruplex formation and enhances its growth inhibitory activity in leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2015-5418

Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data.

BackgroundPausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures.ResultsWe have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present.ConclusionWe discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

Molecular recognition in naphthoquinone derivatives — G-quadruplex complexes by NMR

BackgroundG-quadruplexes have become important drug-design targets for the treatment of various human disorders such as cancer, diabetes and cardiovascular diseases. Recently, G-quadruplex structures have been visualized in the DNA of human cells and appeared to be dynamically sensitive to the cell cycle and stabilized by small molecule ligands. A small library of isoxazolo naphthoquinones (1a–h), which exhibited a strong antiproliferative activity on different cancer cell lines, was studied as potential ligands of G-quadruplex DNA. MethodsThe DNA binding properties of a series of the selected compounds have been analyzed by fluorescence assays. NMR/modeling studies were performed to describe the complexes between G-quadruplex DNA sequences and two selected compounds 1a and 1b. Results1a and 1b in the presence of G-quadruplexes, d(T2AG3T)4, d(TAG3T2A)4 and d(T2G3T2)4, showed good ability of intercalation and the formation of complexes with 2:1 stoichiometry. 1a showed an important interaction with the sequence Pu22 belonging to the promoter of oncogenes c-myc. ConclusionsThe ligands directly interact with the external G-tetrads of the G-quadruplexes, without alterations in the structure of the G-quadruplex core. The role of the adenine moieties over the G-tetrads in the stabilization of the complexes was discussed. General significanceThe results obtained suggested that the strong antiproliferative activity of isoxazolo naphthoquinones is not due to the Hsp90 inhibition, but mainly to the interaction at the level of telomeres and/or at the level of gene promoter. These findings can be used as a basis for the rational drug design of new anticancer agents.

Role of G-quadruplex located at 5ʹ end of mRNAs

BackgroundSecondary structures in 5′ UTR of mRNAs play a critical role in regulating protein synthesis. Though studies have indicated the role of secondary structure G-quadruplex in translational regulation, position-specific effect of G-quadruplex in naturally occurring mRNAs is still not understood. As a pre-initiation complex recognises 5′ cap of the mRNA and scans along the untranslated region (UTR) before initiating translation, the presence of G-quadruplex in 5′ region may have a significant contribution in regulating translation. Here, we investigate the role of G-quadruplex located at the 5′ end of an mRNA. MethodsBiophysical characterisation of putative G-quadruplexes was performed using UV and CD spectroscopy. Functional implication of G-quadruplex in the context of their location was assessed in cellulo using qRT-PCR and dual luciferase assay system. ResultsPG4 sequences in 5′ UTR of AKT interacting protein (AKTIP), cathepsin B (CTSB) and forkhead box E3 (FOXE3) mRNAs form G-quadruplex whereas it is unable to form G-quadruplex in apolipoprotein A-I binding protein (APOA1BP). Our results demonstrated diverse roles of G-quadruplex located at 5′ end of mRNAs. Though G-quadruplex in AKTIP and CTSB mRNA act as inhibitory modules, it activates translation in FOXE3 mRNA. ConclusionsOur works suggests that G-quadruplex present at the 5′ terminal of an mRNA behaves differently in a different gene context. It can activate or inhibit gene expression. General significanceThis study demonstrated that it is difficult to predict the role of G-quadruplex on the basis of its position in 5′ UTR. The neighbouring nucleotide sequence, the intracellular milieu and the interacting partners might render diverse functions to this secondary structure.

Development of an electrochemical insulin sensor based on the insulin-linked polymorphicregion

Here we report the design and fabrication of an electrochemical aptamer-based (E-AB) sensor for detection of insulin. The aptamer used in this study is the insulin-linked polymorphic region (ILPR) sequence, a G-rich sequence that presumably undergoes ligand-induced folding to form a G-quadruplex in presence of insulin. Our circular dichroism data, however, suggests that the ILPR sequence, even in absence of the target, is predominantly in a G-quadruplex-like form. Insulin binding, however, has shown to further induce the formation of the G-quadruplex. To evaluate the potential of the ILPR sequence as a biosensing element, we constructed two E-AB insulin sensors that are identical in all aspects but the location of the methylene blue (MB) redox label. We find that the sensor fabricated with internal MB-modified probes (In-IT) shows enhanced sensing behavior when compared to one fabricated using terminal-MB modified probes (In1). The improvements observed with the In-IT sensor could be attributed to the more effective obstruction of electron transfer upon insulin binding. Overall, both sensors perform well, affording a detection limit of 10nM and 50nM for the In-IT and In1 sensors, respectively.

Mono and Trimethine Cyanines Cyan 40 and Cyan 2 as Probes for Highly Selective Fluorescent Detection of Non-canonical DNA Structures

Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

Triphenylmethane dyes as fluorescent probes for G-quadruplex recognition

Triphenylmethane (TPM) dyes normally render rather weak fluorescence due to easy vibrational deexcitation. However, when they stack onto the two external G-quartets of a G-quadruplex (especially intramolecular G-quadruplex), such vibrations will be restricted, resulting in greatly enhanced fluorescence intensities. Thus, TPM dyes may be developed as sensitive G-quadruplex fluorescent probes. Here, fluorescence spectra and energy transfer spectra of five TPM dyes in the presence of G-quadruplexes, single- or double-stranded DNAs were compared. The results show that the fluorescence spectra of four TPM dyes can be used to discriminate intramolecular G-quadruplexes from intermolecular G-quadruplexes, single- and double-stranded DNAs. The energy transfer fluorescence spectra and energy transfer fluorescence titration can be used to distinguish G-quadruplexes (including intramolecular and intermolecular G-quadruplexes) from single- and double-stranded DNAs. Positive charges and substituent size in TPM dyes may be two important factors in influencing the binding stability of the dyes and G-quadruplexes.

The Human RecQ Helicases, BLM and RECQ1, Display Distinct DNA Substrate Specificities

RecQ helicases maintain chromosome stability by resolving a number of highly specific DNA structures that would otherwise impede the correct transmission of genetic information. Previous studies have shown that two human RecQ helicases, BLM and WRN, have very similar substrate specificities and preferentially unwind noncanonical DNA structures, such as synthetic Holliday junctions and G-quadruplex DNA. Here, we extend this analysis of BLM to include new substrates and have compared the substrate specificity of BLM with that of another human RecQ helicase, RECQ1. Our findings show that RECQ1 has a distinct substrate specificity compared with BLM. In particular, RECQ1 cannot unwind G-quadruplexes or RNA-DNA hybrid structures, even in the presence of the single-stranded binding protein, human replication protein A, that stimulates its DNA helicase activity. Moreover, RECQ1 cannot substitute for BLM in the regression of a model replication fork and is very inefficient in displacing plasmid D-loops lacking a 3'-tail. Conversely, RECQ1, but not BLM, is able to resolve immobile Holliday junction structures lacking an homologous core, even in the absence of human replication protein A. Mutagenesis studies show that the N-terminal region (residues 1-56) of RECQ1 is necessary both for protein oligomerization and for this Holliday junction disruption activity. These results suggest that the N-terminal domain or the higher order oligomer formation promoted by the N terminus is essential for the ability of RECQ1 to disrupt Holliday junctions. Collectively, our findings highlight several differences between the substrate specificities of RECQ1 and BLM (and by inference WRN) and suggest that these enzymes play nonoverlapping functions in cells.

Protection of DNA Ends by Telomeric 3′ G-Tail Sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.