Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A parallel quadruplex DNA is bound tightly but unfolded slowly by pif1 helicase.

ABSTRACT: DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular quadruplex. Pif1 readily unfolds a parallel quadruplex DNA substrate in a multiturnover reaction and also generates some product under single cycle conditions. The rate of ATP hydrolysis by Pif1 is reduced when bound to a parallel quadruplex compared with single-stranded DNA. ATP hydrolysis occurs at a faster rate than quadruplex unfolding, indicating that some ATP hydrolysis events are non-productive during unfolding of intramolecular parallel quadruplex DNA. However, product eventually accumulates at a slow rate.

SUBMITTER: Byrd AK

PROVIDER: S-EPMC4358282 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A parallel quadruplex DNA is bound tightly but unfolded slowly by pif1 helicase.

Byrd Alicia K AK Raney Kevin D KD

The Journal of biological chemistry 20150114 10

DNA sequences that can form intramolecular quadruplex structures are found in promoters of proto-oncogenes. Many of these sequences readily fold into parallel quadruplexes. Here we characterize the ability of yeast Pif1 to bind and unfold a parallel quadruplex DNA substrate. We found that Pif1 binds more tightly to the parallel quadruplex DNA than single-stranded DNA or tailed duplexes. However, Pif1 unwinding of duplexes occurs at a much faster rate than unfolding of a parallel intramolecular q ...[more]

PMID: 25589786

Similar Datasets

DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase.

Project description:G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by noncanonical G-G base pairs. Genome-wide chromatin immunoprecipitation was used to determine the in vivo binding sites of the multifunctional Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high-confidence Pif1-binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, whereas non-G4 Pif1-binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication-stressed Pif1-deficient cells, which was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication, and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.

| S-EPMC3129610 | biostudies-literature

Eukaryotic Pif1 helicase unwinds G-quadruplex and dsDNA using a conserved wedge.

Project description:G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5' single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5' most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.

| S-EPMC11275212 | biostudies-literature

Berenil Binds Tightly to Parallel and Mixed Parallel/Antiparallel G-Quadruplex Motifs with Varied Thermodynamic Signatures.

Project description:Diminazene, DMZ, (or berenil) has been reported as a tight binder of G-quadruplexes. G-Quadruplex structures are often located in the promotor regions of oncogenes and may play a regulatory role in gene expression based on the stability of the folding topology. In this study, attempts have been made to characterize the specificity of DMZ binding toward multiple G-quadruplex topologies or foldamers. Mutant sequences of the G-quadruplex forming promotor regions of several oncogenes were designed to exhibit restricted loop lengths and folding topologies. Circular dichroism was used to confirm the quadruplex topology of mutant BCL2, KRAS, and c-MYC sequences, human telomere (Na+ and K+) G-quadruplexes and their complexes with DMZ and analogs thereof. Isothermal titration calorimetry was used to generate a complete thermodynamic profile (ΔG, ΔH, -TΔS) for the formation of DMZ and analog complexes with the target G-quadruplexes. DMZ binds to parallel and/or mixed parallel/antiparallel quadruplex DNA motifs with stoichiometries up to 8:1 and via three binding modes with varying affinities. In the case of the parallel G-quadruplexes, with the exception of the long-looped c-MYC mutant, the highest affinity binding event (mode 1) is driven by enthalpy. DMZ binding to the long-looped c-MYC mutant exhibits a very favorable entropy change in addition to a moderately favorable enthalpy change. Mode 1 binding to the antiparallel and mixed parallel/antiparallel hTel quadruplexes is also driven by favorable enthalpy changes. In all cases, the intermediate DMZ affinity binding (mode 2) is driven almost entirely by entropy, with small or unfavorable enthalpic contributions. The weakest binding event (mode 3) is also entropically driven with small or moderate enthalpic contributions.

| S-EPMC6173502 | biostudies-literature

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family.

Project description:The two PIF1 family helicases in Saccharomyces cerevisiae, Rrm3, and ScPif1, associate with thousands of sites throughout the genome where they perform overlapping and distinct roles in telomere length maintenance, replication through non-histone proteins and G4 structures, lagging strand replication, replication fork convergence, the repair of DNA double-strand break ends, and transposable element mobility. ScPif1 and its fission yeast homolog Pfh1 also localize to mitochondria where they protect mitochondrial genome integrity. In addition to yeast serving as a model system for the rapid functional evaluation of human Pif1 variants, yeast cells lacking Rrm3 have proven useful for elucidating the cellular response to replication fork pausing at endogenous sites. Here, we review the increasingly important cellular functions of the yeast PIF1 helicases in maintaining genome integrity, and highlight recent advances in our understanding of their roles in facilitating fork progression through replisome barriers, their functional interactions with DNA repair, and replication stress response pathways.

| S-EPMC7073672 | biostudies-literature

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks.

Project description:Pif-1 proteins are 5'-->3' superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3' ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.

| S-EPMC2770657 | biostudies-literature

The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.

Project description:In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

| S-EPMC2673046 | biostudies-literature

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA.

Project description:In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its functions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA unwinding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 5' to 3' directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 5' to 3' directionality.

| S-EPMC3632115 | biostudies-literature

Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress.

Project description:Unwinding duplex DNA is a critical processing step during replication, repair and transcription. Pif1 are highly conserved non-processive 5'->3' DNA helicases with well-established roles in maintenance of yeast genome stability. However, the function of the sole member of Pif1 family in humans remains unclear. Human PIF1 is essential for tumour cell viability, particularly during replication stress, but is dispensable in non-cancerous cells and Pif1 deficient mice. Here we report that suppression of PIF1 function slows replication fork rates and increases arrested forks during normal cycling conditions. Importantly, PIF1-dependent replication impediments impair S-phase progression and reduce proliferation rates of RAS oncogene-transformed fibroblasts, where replication fork slowing is exacerbated, but not parental, non-cancerous cells. Disrupted fork movement upon PIF1-depletion does not enhance double-stranded break formation or DNA damage responses but affects resumption of DNA synthesis after prolonged replication inhibitor exposure, accompanied by diminished new origin firing and mainly S-phase entry. Taken together, we characterised a functional role for human PIF1 in DNA replication that becomes important for cell growth under oncogenic stress. Given that oncogenes induce high levels of replication stress during the early stages of tumorigenesis, this function of PIF1 could become critical during cancer development.

| S-EPMC4294361 | biostudies-literature

Structure and function of Pif1 helicase.

Project description:Pif1 family helicases have multiple roles in the maintenance of nuclear and mitochondrial DNA in eukaryotes. Saccharomyces cerevisiae Pif1 is involved in replication through barriers to replication, such as G-quadruplexes and protein blocks, and reduces genetic instability at these sites. Another Pif1 family helicase in S. cerevisiae, Rrm3, assists in fork progression through replication fork barriers at the rDNA locus and tRNA genes. ScPif1 (Saccharomyces cerevisiae Pif1) also negatively regulates telomerase, facilitates Okazaki fragment processing, and acts with polymerase ? in break-induced repair. Recent crystal structures of bacterial Pif1 helicases and the helicase domain of human PIF1 combined with several biochemical and biological studies on the activities of Pif1 helicases have increased our understanding of the function of these proteins. This review article focuses on these structures and the mechanism(s) proposed for Pif1's various activities on DNA.

| S-EPMC5870758 | biostudies-literature

Pif1 is a force-regulated helicase.

Project description:Pif1 is a prototypical member of the 5' to 3' DNA helicase family conserved from bacteria to human. It has a high binding affinity for DNA, but unwinds double-stranded DNA (dsDNA) with a low processivity. Efficient DNA unwinding has been observed only at high protein concentrations that favor dimerization of Pif1. In this research, we used single-molecule fluorescence resonance energy transfer (smFRET) and magnetic tweezers (MT) to study the DNA unwinding activity of Saccharomyces cerevisiae Pif1 (Pif1) under different forces exerted on the tails of a forked dsDNA. We found that Pif1 can unwind the forked DNA repetitively for many unwinding-rezipping cycles at zero force. However, Pif1 was found to have a very limited processivity in each cycle because it loosened its strong association with the tracking strand readily, which explains why Pif1 cannot be observed to unwind DNA efficiently in bulk assays at low protein concentrations. The force enhanced the unwinding rate and the total unwinding length of Pif1 significantly. With a force of 9 pN, the rate and length were enhanced by more than 3- and 20-fold, respectively. Our results imply that the DNA unwinding activity of Pif1 can be regulated by force. The relevance of this characteristic of Pif1 to its cellular functions is discussed.

| S-EPMC4872122 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data