Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity

2010 ◽  
Vol 430 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Cyril M. Sanders
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Genome Stability ◽  
Dna Helicase ◽  
Dna Unwinding ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
G Quadruplex ◽  
Pif1 Helicase

Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206–620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5′ ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.

scholarly journals ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yu-Ching Teng ◽  
Aishwarya Sundaresan ◽  
Ryan O’Hara ◽  
Vincent U. Gant ◽  
Minhua Li ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
Heterochromatin Formation ◽  
Replicative Stress ◽  
G4 Dna ◽  
Mutation Burden ◽  
G Quadruplex ◽  
Dna Replication Stress

AbstractATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with a number of the MCM replication complex subunits and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

scholarly journals ATRX promotes heterochromatin formation to protect cells from G-quadruplex DNA-mediated stress

2021 ◽  
Author(s):  
Yu-Ching Teng ◽  
Aishwarys Sundaresan ◽  
Ryan O'Hara ◽  
Vincent U Gant ◽  
Minhua Li ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Stability ◽  
Helicase Domain ◽  
Quadruplex Dna ◽  
Heterochromatin Formation ◽  
Replicative Stress ◽  
G4 Dna ◽  
Mutation Burden ◽  
G Quadruplex ◽  
Dna Replication Stress

ATRX is a tumor suppressor that has been associated with protection from DNA replication stress, purportedly through resolution of difficult-to-replicate G-quadruplex (G4) DNA structures. While several studies demonstrate that loss of ATRX sensitizes cells to chemical stabilizers of G4 structures, the molecular function of ATRX at G4 regions during replication remains unknown. Here, we demonstrate that ATRX associates with the MCM replication complex and that loss of ATRX leads to G4 structure accumulation at newly synthesized DNA. We show that both the helicase domain of ATRX and its H3.3 chaperone function are required to protect cells from G4-induced replicative stress. Furthermore, these activities are upstream of heterochromatin formation mediated by the histone methyltransferase, ESET, which is the critical molecular event that protects cells from G4-mediated stress. In support, tumors carrying mutations in either ATRX or ESET show increased mutation burden at G4-enriched DNA sequences. Overall, our study provides new insights into mechanisms by which ATRX promotes genome stability with important implications for understanding impacts of its loss on human disease.

scholarly journals G-rich telomeric and ribosomal DNA sequences from the fission yeast genome form stable G-quadruplex DNA structuresin vitroand are unwound by the Pfh1 DNA helicase

2016 ◽  
Vol 44 (13) ◽  
pp. 6213-6231 ◽  
Author(s):  
Marcus Wallgren ◽  
Jani B. Mohammad ◽  
Kok-Phen Yan ◽  
Parham Pourbozorgi-Langroudi ◽  
Mahsa Ebrahimi ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Ribosomal Dna ◽  
Dna Sequences ◽  
Dna Helicase ◽  
Yeast Genome ◽  
Quadruplex Dna ◽  
G Quadruplex

scholarly journals Pif1 helicase unfolding of G-quadruplex DNA is highly dependent on sequence and reaction conditions

2018 ◽  
Vol 293 (46) ◽  
pp. 17792-17802 ◽  
Author(s):  
Alicia K. Byrd ◽  
Matthew R. Bell ◽  
Kevin D. Raney
Keyword(s):  
Dna Sequences ◽  
Atp Hydrolysis ◽  
Monovalent Cation ◽  
Product Formation ◽  
Telomeric Dna ◽  
Reaction Conditions ◽  
Specific Sequence ◽  
Quadruplex Dna ◽  
G Quadruplex ◽  
Pif1 Helicase

In addition to unwinding double-stranded nucleic acids, helicase activity can also unfold noncanonical structures such as G-quadruplexes. We previously characterized Pif1 helicase catalyzed unfolding of parallel G-quadruplex DNA. Here we characterized unfolding of the telomeric G-quadruplex, which can fold into antiparallel and mixed hybrid structures and found significant differences. Telomeric DNA sequences are unfolded more readily than the parallel quadruplex formed by the c-MYC promoter in K+. Furthermore, we found that under conditions in which the telomeric quadruplex is less stable, such as in Na+, Pif1 traps thermally melted quadruplexes in the absence of ATP, leading to the appearance of increased product formation under conditions in which the enzyme is preincubated with the substrate. Stable telomeric G-quadruplex structures were unfolded in a stepwise manner at a rate slower than that of duplex DNA unwinding; however, the slower dissociation from G-quadruplexes compared with duplexes allowed the helicase to traverse more nucleotides than on duplexes. Consistent with this, the rate of ATP hydrolysis on the telomeric quadruplex DNA was reduced relative to that on single-stranded DNA (ssDNA), but less quadruplex DNA was needed to saturate ATPase activity. Under single-cycle conditions, telomeric quadruplex was unfolded by Pif1, but for the c-MYC quadruplex, unfolding required multiple helicase molecules loaded onto the adjacent ssDNA. Our findings illustrate that Pif1-catalyzed unfolding of G-quadruplex DNA is highly dependent on the specific sequence and the conditions of the reaction, including both the monovalent cation and the order of addition.

scholarly journals Yeast transcription co-activator Sub1 and its human homolog PC4 preferentially bind to G-quadruplex DNA

2015 ◽  
Vol 51 (33) ◽  
pp. 7242-7244 ◽  
Author(s):  
Jun Gao ◽  
Boris L. Zybailov ◽  
Alicia K. Byrd ◽  
Wezley C. Griffin ◽  
Shubeena Chib ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Human Homolog ◽  
Dna Metabolism ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
G Quadruplex

DNA binding proteins Sub1 and PC4 preferentially bind to G-quadruplex DNA, providing a new link between DNA metabolism and G4-DNA.

scholarly journals Replication of G Quadruplex DNA

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 95 ◽  
Author(s):  
Leticia Koch Lerner ◽  
Julian E. Sale
Keyword(s):  
Dna Replication ◽  
Dna Sequences ◽  
Secondary Structures ◽  
Dna Unwinding ◽  
Chromosomal Dna ◽  
Quadruplex Dna ◽  
Epigenetic Instability ◽  
G Quadruplex ◽  
Dna Secondary Structures ◽  
Dna Template

A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

scholarly journals The WYL domain of the PIF1 helicase from the thermophilic bacteriumThermotoga elfiiis an accessory single-stranded DNA binding module

2017 ◽  
Author(s):  
Nicholas M. Andis ◽  
Christopher W. Sausen ◽  
Ashna Alladin ◽  
Matthew L. Bochman
Keyword(s):  
Unknown Function ◽  
Genome Stability ◽  
Dna Helicase ◽  
Thermophilic Bacterium ◽  
Helicase Domain ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Pif1 Helicase

ABSTRACTPIF1 family helicases are conserved from bacteria to man. With the exception of the well-studied yeast PIF1 helicases (e.g., ScPif1 and ScRrm3), however, very little is known about how these enzymes help maintain genome stability. Indeed, we lack a basic understanding of the protein domains found N- and C-terminal to the characteristic central PIF1 helicase domain in these proteins. Here, using chimeric constructs, we show that the ScPif1 and ScRrm3 helicase domains are interchangeable and that the N-terminus of ScRrm3 is important for its functionin vivo. This suggests that PIF1 family helicases evolved functional modules fused to a generic motor domain. To investigate this hypothesis, we characterized the biochemical activities of the PIF1 helicase from the thermophilic bacteriumThermotoga elfii(TePif1), which contains a C-terminal WYL domain of unknown function. Like helicases from other thermophiles, recombinant TePif1 was easily prepared, thermostablein vitro, and displayed activities similar to its eukaryotic homologs. We also found that the WYL domain was necessary for high-affinity single-stranded DNA (ssDNA) binding and affected both ATPase and helicase activities. Deleting the WYL domain from TePif1 or mutating conserved residues in the predicted ssDNA binding site uncoupled ATPase activity and DNA unwinding, leading to higher rates of ATP hydrolysis but less efficient DNA helicase activity. Our findings suggest that the domains of unknown function found in eukaryotic PIF1 helicases may also confer functional specificity and additional activities to these enzymes, which should be investigated in future work.

scholarly journals Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jose F Moruno-Manchon ◽  
Pauline Lejault ◽  
Yaoxuan Wang ◽  
Brenna McCauley ◽  
Pedram Honarpisheh ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Memory Deficits ◽  
Dna Helicase ◽  
Vitro Assay ◽  
Dna Structures ◽  
First Intron ◽  
G4 Dna ◽  
G Quadruplex ◽  
Dna Stabilization

Guanine-rich DNA sequences can fold into four-stranded G-quadruplex (G4-DNA) structures. G4-DNA regulates replication and transcription, at least in cancer cells. Here, we demonstrate that, in neurons, pharmacologically stabilizing G4-DNA with G4 ligands strongly downregulates the Atg7 gene. Atg7 is a critical gene for the initiation of autophagy that exhibits decreased transcription with aging. Using an in vitro assay, we show that a putative G-quadruplex-forming sequence (PQFS) in the first intron of the Atg7 gene folds into a G4. An antibody specific to G4-DNA and the G4-DNA-binding protein PC4 bind to the Atg7 PQFS. Mice treated with a G4 stabilizer develop memory deficits. Brain samples from aged mice contain G4-DNA structures that are absent in brain samples from young mice. Overexpressing the G4-DNA helicase Pif1 in neurons exposed to the G4 stabilizer improves phenotypes associated with G4-DNA stabilization. Our findings indicate that G4-DNA is a novel pathway for regulating autophagy in neurons.

scholarly journals Subtle structural alterations in G-quadruplex DNA regulate site specificity of fluorescence light-up probes

2020 ◽  
Vol 48 (3) ◽  
pp. 1108-1119 ◽  
Author(s):  
Rajendra Kumar ◽  
Karam Chand ◽  
Sudipta Bhowmik ◽  
Rabindra Nath Das ◽  
Snehasish Bhattacharjee ◽  
...  
Keyword(s):  
Rational Design ◽  
Dna Structure ◽  
Biological Processes ◽  
Chemical Research ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Future Studies ◽  
G4 Dna ◽  
G Quadruplex ◽  
Fluorescence Light

Abstract G-quadruplex (G4) DNA structures are linked to key biological processes and human diseases. Small molecules that target specific G4 DNA structures and signal their presence would therefore be of great value as chemical research tools with potential to further advance towards diagnostic and therapeutic developments. However, the development of these types of specific compounds remain as a great challenge. In here, we have developed a compound with ability to specifically signal a certain c-MYC G4 DNA structure through a fluorescence light-up mechanism. Despite the compound's two binding sites on the G4 DNA structure, only one of them result in the fluorescence light-up effect. This G-tetrad selectivity proved to originate from a difference in flexibility that affected the binding affinity and tilt the compound out of the planar conformation required for the fluorescence light-up mechanism. The intertwined relation between the presented factors is likely the reason for the lack of examples using rational design to develop compounds with turn-on emission that specifically target certain G4 DNA structures. However, this study shows that it is indeed possible to develop such compounds and present insights into the molecular details of specific G4 DNA recognition and signaling to advance future studies of G4 biology.

scholarly journals G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

2018 ◽  
Author(s):  
Yuxiang Wang ◽  
Jie Yang ◽  
Wei Wu ◽  
Rachna Shah ◽  
Carla Danussi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Malignant Glioma ◽  
Model Systems ◽  
Copy Number Alterations ◽  
Molecular Alteration ◽  
Quadruplex Dna ◽  
G4 Dna ◽  
G Quadruplex

AbstractMutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

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