The unimolecular forms often occur naturally near the ends of the chromo. The essential unit of g4 dna is the gquartet, a planar array of guanines stabilized by hoogsteen base pairing between the n7 group of one guanine and the extracyclic amino group of its neighbor. Insights into gquadruplexinteractive small molecule drug. Saccharomyces cerevisiae pif1 is involved in replication through barriers to replication, such as gquadruplexes and protein blocks, and reduces genetic instability at these sites. Inhibition of the blooms and werners syndrome helicases. Gquadruplex unwinding helicases and their function in vivo. Gquadruplex dna presents a potential target for the design and development of novel anticancer drugs. Myc is a critical oncogene with a g4 in its promoter mycg4, which acts as a transcription silencer. In molecular biology, gquadruplex secondary structures g4 are formed in nucleic acids by sequences that are rich in guanine. For example, pif1 helicase has been shown to act specifically at formed g4 structures during dna replication, whereas the recq helicases are. Bohr laboratory of molecular gerontology, national institute on aging, nih, 5600 nathan shock drive, baltimore, md 21224, usa. Even though g4 dna is not a substrate for all helicases, both yeast sgs1p and its close human homolog, blm, unwind g4 dna with at least 15. Specific interactions of distamycin with gquadruplex dna. Gquadruplexes and their regulatory roles in biology pdf.
Shiels and nancy maizels1,2 1department of biochemistry university of washington medical school, seattle, wa. Crystal structure of parallel quadruplexes from human telomeric dna. For comparison, we also tested the inhibition of g4 ceb1 unwinding by nmm, reported to be a potent inhibitor of g4 dna unwinding by the escherichia coli recq helicase and its eukaryotic homologues sgs1 and blm helicases 35. We reveal herein that ddx5, a founding member of the deadbox rna helicase family, can unfold dna g4s. Molecules free fulltext gquadruplex structures in the. Gquadruplexes are secondary structures that may form within guaninerich nucleic acid sequences. Dna structures will lead to the discovery of novel molecular entities that bind not only to g4.
Therefore, to achieve therapeutic selectivity using gquadruplexes as targets for drug design, it will be necessary to differentiate. How recq family helicases recognize this substrate has not been established. Structurebased design of selective and potent g quadruplexmediated telomerase inhibitors. The g4 dna substrate tested was formed from the tp oligonucleotide, which carries a consensus sequence from the murine immunoglobulin s. For comparison, we also tested the inhibition of g4ceb1 unwinding by nmm, reported to be a potent inhibitor of g4dna unwinding by the escherichia coli recq helicase and its eukaryotic homologues sgs1 and blm helicases 35. G4 dna presents obstacles for the replication machinery, and both eukaryotic dna helicases and polymerases have evolved to resolve and copy g4 dna in vivo. Jul 26, 2016 gquadruplex gq is a fourstranded dna structure that forms in the human genome and influences gene expression and dna replication.
Author summary common fragile sites cfss are large chromosomal regions which are more prone to breakage than other places in the genome. In addition, g4 forming sequences are prevalent in gene promoters, suggesting that g4 resolving helicases act to modulate transcription. Frontiers structural mechanisms of human recq helicases. Three conformational snapshots of the hepatitis c virus. Considerable circumstantial evidence suggests that these structures can exist in vivo in specific regions of the genome including the telomeric ends of chromosomes and. Many gqresolving helicases have a higher affinity toward gq dna than to other forms of singlestrand ss or double strand ds dna. Human premature aging, dna repair and recq helicases. The interplay between gquadruplex and transcription. The unimolecular forms often occur naturally near the ends of the chromosomes, better known as the telomeric regions, and in transcriptional regulatory. Guaninerich dna sequences can fold into fourstranded, noncanonical secondary structures called gquadruplexes g4s. Understanding how cfss are protected to prevent genome. Gquadruplexes and their regulatory roles in biology gquadruplexes and their regulatory roles in biology. However, due to the location of gquadruplex motifs. Quadruplexes are fourstranded dna or rna structures formed from tandem repeats of guaninerich sequences.
Welldesigned studies in caenorhabditis elegans showed that. We now report that the blm helicase can unwind g4 dna. Human premature aging, dna repair and recq helicases core. Understanding the roles of g4 resolving dna helicases may lead to new insights for the design of g4 ligands that block g4 helicases or related proteins, thereby interfering with telomere semiconservative replication or impeding telomerasecatalysed elongation in cancer cells and causing them to senesce fig. Identification of gquadruplexbinding inhibitors of myc. Previous studies have identified a series of trisubstituted acridine derivatives that are potent and selective ligands for gquadruplex dna. The g4 stabilising ligand pyridostatin generates dna doublestrand breaks. New helicase assays that recognise therapeutically important g4. Pdf guaninerich dna strands can fold in vitro into noncanonical dna structures called gquadruplexes. Considerable circumstantial evidence suggests that these structures can exist in vivo in specific regions of the genome including the telomeric ends of chromosomes and oncogene.
G4s were initially considered a structural curiosity, but recent evidence suggests their involvement in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, together with numerous connections to cancer biology. Therefore, to achieve therapeutic selectivity using gquadruplexes as targets for drug design, it will be necessary to differentiate between. Although mice deficient in these helicases are indistinguishable. The concept that gquadruplex g4 structures can form within dna or rna in vitro has been long known and extensively discussed. Only a handful of these helicases are known to preferentially bind and unwind g. Crystal structure of parallel quadruplexes from human telomeric. Apr 15, 2001 escherichia coli recq helicase unwinds g4 and g2. Dna helicases implicated in chromosomal instability disorders as targets for the discovery nucleic acids research. Quadruplex g4 dna structures are one of the most stable non. 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. To counteract such an effect, cells possess special helicases dedicated to unfolding gq structures. These ligands were shown to selectively exert an antiproliferative and. Gquadruplexes g4 are 4stranded nucleic acid secondary structures.
Strikingly, g4 dna is a preferred substrate of the blm helicase, as measured both by efficiency of unwinding and by competition. Apr 23, 2019 dhx36 also called rhau and g4r1 is an rna helicase that can resolve gquadruplex g4 dna and g4rna with high specificity and affinity 1,2,3,4,5,6. Molecular recognition of the hybridtype gquadruplexes in. Viral and cellular rna helicases as antiviral targets. A conserved g4 dna binding domain in recq family helicases michael d. Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial dna in eukaryotes. Mycobacterium tuberculosis ding is a structurespecific. They are expressed highly in rapidly proliferating cells and tumor cells, suggesting that they have important roles in the replication of a genome. Gquadruplex unwinding helicases and their function in.
Although g4 dna and potential involvement of dna helicases will be. Trf2 overexpression might counteract the deleterious effects of the stabilization of g4 dna by rhps4 through several means, including the formation of tloop that would mask the 3. Wrn and blm are distinguished from other helicases by. Jul 24, 2014 mycobacterium tuberculosis ding is a structurespecific helicase that unwinds g4 dna. Telomeres have received much attention in this regard since they can fold into several distinct intramolecular gquadruplexes, leading to the rational design and development of gquadruplex. Gquadruplexes and helicases nucleic acids research oxford. Rationale for clinical evaluation of tarloxotinib bromide, a. Our findings indicate that g4dna is a novel pathway for. Helicases catalytically unwind duplex dna or rna using energy derived from the hydrolysis of nucleoside triphosphates and are attractive drug targets because they are required for viral replication. Dnas, and drug design hale, tracy k norris, gillian e jameson, geoffrey b filichev, vyacheslav v.
We identify the rqc domain, found only in recq family enzymes, as an independent, high affinity and conserved g4 dna binding domain. Dna quadruplexes, also known as gquadruplex structures, comprise stacked tetrads, each of which arises from the planar association of four guanines in a cyclic hoogsteen hydrogenbonding arrangement. Quadruplexes are thermodynamically stable structures and once formed can be a hindrance to cellular processes. Structure and function of pif1 helicase biochemical. Mycg4 is very stable and the pathological activation of myc in cancers requires its active unfolding.
Linking these two activities telomerase and g4 will certainly be an interesting development of future molecular genetic studies of pif1. The blm g4 dna unwinding activity is atpdependent and requires a short 3. The malaria parasite expresses different versions of its surface epitopes, allowing it to evade detection by the host immune system. The recent cocrystal structure of dhx36 bound g4dna revealed an intimate contact, but did not. In recent years, accumulating evidences imply that gquadruplex structures form in vivo. Blm, the gene that is defective in blooms syndrome, encodes a protein homologous to recq subfamily helicases that functions as a 3. Here, we show that g4 dna is a preferred target for blm helicase within the context of long dna molecules. We identify the rqc domain, found only in recq family enzymes, as an independent, high af. Crystal structure of parallel quadruplexes from human. However, due to the location of gquadruplex motifs and their. They are helical in shape and contain guanine tetrads that can form from one, two or four strands. G4 dna, transcription, rloops, top1, supercoiling, genome stability. Our results show that the ability to unwind g4 dna is not a universal property of dna helicases. Filichev1 1institute of fundamental sciences, massey university, palmerston north, new zealand the human genome contains nearly 100 molecular motors that unwind nucleic acids.
Rationale for clinical evaluation of tarloxotinib bromide. Human premature aging, dna repair and recq helicases robert m. Singlemolecule imaging reveals a common mechanism shared. Not all helicases can unwind g4 dnaspecific helicases are associated with replication and recombination, but these helicases could mitigate against formation of gdna only if they can unwind this substrate efficiently. G4 dna motifs might play an important role in this process because they facilitate recombination and subsequently. Structure and function of pif1 helicase biochemical society.
Overexpressing the g4dna helicase pif1 in neurons exposed to the g4 stabilizer improves phenotypes associated with g4dna stabilization. Selective interactions of cationic porphyrins with g. Thus, human telomeric gquadruplexes are considered attractive targets for anticancer drugs. Dna helicases involved in dna repair and their roles in cancer. Strikingly, nmm was fairly inefficient in inhibiting g4 dna unwinding by pif1. Unraveling the importance of the malaria parasite helicases. Substratespecific inhibition of recq helicase nucleic. Us7312082b2 crystal structure of gquadruplex containing. Although mice deficient in these helicases are indistinguishable from.
Jci telomere damage induced by the gquadruplex ligand. We reveal herein that ddx5, a founding member of the deadbox rna helicase family, can unfold. These ligands have been shown previously to inhibit the activity of telomerase, the specialized. G4 motifs confer the ability to form a g4 dna structure, also known as a quadruplex by analogy with the bform dna duplex. One strategy for inhibition of myc expression is through stabilization of the gquadruplex g4, a grich dna secondary structure found within the myc promoter. Genetic instability triggered by gquadruplex interacting. In our study, we examined three such gqresolving helicases, using single. Choudhary midna helicases implicated in chromosomal instability disorders as targets for the discovery of novel anticancer agentsfrontiers in drug design and discovery 2007. To investigate the mechanism of hcv helicase action, we determined the structures of ns3h bound to ssdna da6 or dt6 alone and in combination with groundstate and transitionstate mimics of atp fig. Dhx36 is a deahbox helicase that resolves parallel gquadruplex structures formed in dna and rna.
Initially, inefficient regulation of gquadruplex structures was mainly associated with genome instability. They are a part of normal chromosome structure and are present in all human beings, but are also hotspots for chromosomal rearrangement during oncogenesis. Dna helicases use the energy of atp hydrolysis to disrupt dna base pairing and displace proteins from dna in order to. Rna gquadruplex is resolved by repetitive and atpdependent. Ddx5 helicase resolves gquadruplex and is involved in myc.
Request pdf helicases, g4dnas, and drug design new helicase assays that recognise therapeutically important g4dna structures will lead to the discovery of. Mms1 is an assistant for regulating gquadruplex dna structures. They are present throughout the genome, namely at telomeres, in promoter and untranslated regions of important genes. During the last decade, fourstranded helical structures called gquadruplex or g4 have emerged from being a structural curiosity observed in vitro, to being recognized as a possible nucleic acid based mechanism for regulating multiple biological. Rna immunoprecipitation ripchip analysis identified approximately 100 rnas associated with rhau in vivo and the majority contained pg4s. Although it might be tempting to inhibit oligomerization. G4s were initially considered a structural curiosity, but recent evidence suggests their involvement in key genome functions such as transcription, replication, genome stability, and epigenetic regulation, together with numerous connections to cancer.
A conserved g4 dna binding domain in recq family helicases. In humans, mutations in these proteins lead to rare genetic diseases associated with cancer predisposition and accelerated aging. Mycobacterium tuberculosis ding is a structurespecific helicase that unwinds g4 dna. The present invention relates to a crystal structure of gquadruplexes and its use. They are helical structures containing guanine tetrads that can form from one, two or four strands. The requirement for helicases to carry out roles in dna repair through their catalytic functions or interactions with other proteins continues to be an important area of study. Frontiers structural mechanisms of human recq helicases wrn. These results suggest that g4 dna may be a natural substrate of blm in vivo and that the failure to unwind g4 dna may cause the genomic instability and increased frequency of sister chromatid exchange characteristic. Recql1 and wrn helicases in the human recq helicase family participate in maintaining genome stability, dna repair, replication, and recombination pathways in the cell cycle. In recent years, the gquadruplex field has blossomed as new evidence strongly suggests that such alternately folded dna structures are likely to exist in vivo. Gquadruplex gq is a fourstranded dna structure that forms in the human genome and influences gene expression and dna replication. Since then, the number of studies reporting gquadruplex dna unfolding by helicase. Gquadruplex g4 structures can form in dna and rna molecules when.
Stably formed gq structures can act as a physical blockade to disrupt genomic processes. G4 dna is a noncanonical dna structure consisting of a stacked array of gquartets held together by base pairing between guanine bases. Although they differ in directionality and substrate specificity, it is not clear whether these helicases have an overlapping or complementary role in resolving gq dna structures. Gquadruplex gq is a four stranded dna secondary structure that arises from a guanine rich sequence. Roshan singh thakur from the department of biochemistry, indian institute of science, bangalore 560012, india. The recq family dna helicases werner syndrome protein wrn and bloom syndrome protein blm play a key role in protecting the genome against deleterious changes. Dna helicases use the energy of atp hydrolysis to disrupt dna base pairing and. The invention provides a crystal of an intramolecular gquadruplex structure having a hexagonal space group p6, and unit cell dimensions ab56. If gquadruplexes form so readily in vitro, nature will have found a way of using them in vivo statement by aaron klug over 30 years ago. Because gquadruplex dna exhibits structural polymorphism, different gquadruplex typologies may be associated with different cellular processes. Wrn and blm are distinguished from other helicases by possessing signature tandem domains toward the c. We focus the discussion largely on the importance of gquadruplexes. The formation of g4 dna requires a cluster of guanineruns within a strand of dna.
Inhibition of the blooms and werners syndrome helicases by. Singlemolecule imaging reveals a common mechanism shared by. In addition to the familiar duplex dna, certain dna sequences can fold into secondary structures that are fourstranded. Jul 26, 2016 gquadruplex gq is a four stranded dna secondary structure that arises from a guanine rich sequence. Rhau is a deahbox helicase that exhibits g4rna and g4dna binding and resolving activity. Dna helicases as targets for anticancer drugs request pdf. Gquadruplex g4 dna secondary structures formed in human telomeres have been shown to inhibit cancerspeci. Electrostatic analysis of the hcv helicase shows both activesite and nonactivesite locations 73 that could be exploited for drug design. Crick structures that can form within guaninerich dna sequences such as telomeric repeats. Request pdf helicases, g4dnas, and drug design new helicase assays that recognise therapeutically important g4dna structures will lead to the discovery of novel molecular entities that bind. Oct 08, 2019 gquadruplexes g4 are 4stranded nucleic acid secondary structures. Hale institute of fundamental sciences, massey university, private bag 11 222, palmerston north new zealand, fax. The myc oncogene is overexpressed in many cancers, yet targeting it for cancer therapy has remained elusive.
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