Browse
Submit Data
Databases
API
Help

Dataset Information

28 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain.

ABSTRACT: : Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1?N) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1?N was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.

SUBMITTER: Nickens DG

PROVIDER: S-EPMC6628001 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Direct quantification of the translocation activities of Saccharomyces cerevisiae Pif1 helicase.

Project description:Saccharomyces cerevisiae Pif1 (ScPif1) is known as an ATP-dependent DNA helicase that plays critical roles in a number of important biological processes such as DNA replication, telomere maintenance and genome stability maintenance. Besides its DNA helicase activity, ScPif1 is also known as a single-stranded DNA (ssDNA) translocase, while how ScPif1 translocates on ssDNA is unclear. Here, by measuring the translocation activity of individual ScPif1 molecules on ssDNA extended by mechanical force, we identified two distinct types of ssDNA translocation. In one type, ScPif1 moves along the ssDNA track with a rate of ∼140 nt/s in 100 μM ATP, whereas in the other type, ScPif1 is immobilized to a fixed location of ssDNA and generates ssDNA loops against force. Between the two, the mobile translocation is the major form at nanomolar ScPif1 concentrations although patrolling becomes more frequent at micromolar concentrations. Together, our results suggest that ScPif1 translocates on extended ssDNA in two distinct modes, primarily in a 'mobile' manner.

| S-EPMC6698741 | biostudies-literature

Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain.

Project description:The evolutionary conserved PIF1 DNA helicase family appears to have largely nonoverlapping cellular functions. To better understand the functions of human PIF1, we investigated biochemical properties of this protein. Analysis of single-stranded (ss) DNA-dependent ATPase activity revealed nonstructural ssDNA to greatly stimulate ATPase activity due to a high affinity for PIF1, even though PIF1 preferentially unwinds forked substrates. This suggests that PIF1 needs a ssDNA region for loading and a forked structure for translocation entrance into a double strand region. Deletion analysis demonstrated novel functions of a unique N-terminal portion, named the PIF1 N-terminal (PINT) domain. When the PINT domain was truncated, apparent affinity for ssDNA and unwinding activity were much reduced, even though the maximum velocity of ATPase activity and K(m) value for ATP were not affected. We suggest that the PINT domain contributes to enhancing the interaction with ssDNA through intrinsic binding activity. In addition, we found DNA strand-annealing activity, also residing in the PINT domain. Notably, the unwinding and annealing activities were inhibited by replication protein A. These results suggest that the functions of PIF1 might be restricted with particular situations and DNA structures.

| S-EPMC2577353 | 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

Identification of the nuclear localization signal in the Saccharomyces cerevisiae Pif1 DNA helicase.

Project description:Saccharomyces cerevisiae Pif1 is a multi-functional DNA helicase that plays diverse roles in the maintenance of the nuclear and mitochondrial genomes. Two isoforms of Pif1 are generated from a single open reading frame by the use of alternative translational start sites. The Mitochondrial Targeting Signal (MTS) of Pif1 is located between the two start sites, but a Nuclear Localization Signal (NLS) has not been identified. Here we used sequence and functional analysis to identify an NLS element. A mutant allele of PIF1 (pif1-NLSΔ) that lacks four basic amino acids (781KKRK784) in the carboxyl-terminal domain of the 859 amino acid Pif1 was expressed at wild type levels and retained wild type mitochondrial function. However, pif1-NLSΔ cells were defective in four tests for nuclear function: telomere length maintenance, Okazaki fragment processing, break-induced replication (BIR), and binding to nuclear target sites. Fusing the NLS from the simian virus 40 (SV40) T-antigen to the Pif1-NLSΔ protein reduced the nuclear defects of pif1-NLSΔ cells. Thus, four basic amino acids near the carboxyl end of Pif1 are required for the vast majority of nuclear Pif1 function. Our study also reveals phenotypic differences between the previously described loss of function pif1-m2 allele and three other pif1 mutant alleles generated in this work, which will be useful to study nuclear Pif1 functions.

| S-EPMC10399864 | biostudies-literature

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

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

Biochemical studies of the Saccharomyces cerevisiae Mph1 helicase on junction-containing DNA structures.

Project description:Saccharomyces cerevisiae Mph1 is a 3-5' DNA helicase, required for the maintenance of genome integrity. In order to understand the ATPase/helicase role of Mph1 in genome stability, we characterized its helicase activity with a variety of DNA substrates, focusing on its action on junction structures containing three or four DNA strands. Consistent with its 3' to 5' directionality, Mph1 displaced 3'-flap substrates in double-fixed or equilibrating flap substrates. Surprisingly, Mph1 displaced the 5'-flap strand more efficiently than the 3' flap strand from double-flap substrates, which is not expected for a 3-5' DNA helicase. For this to occur, Mph1 required a threshold size (>5 nt) of 5' single-stranded DNA flap. Based on the unique substrate requirements of Mph1 defined in this study, we propose that the helicase/ATPase activity of Mph1 play roles in converting multiple-stranded DNA structures into structures cleavable by processing enzymes such as Fen1. We also found that the helicase activity of Mph1 was used to cause structural alterations required for restoration of replication forks stalled due to damaged template. The helicase properties of Mph1 reported here could explain how it resolves D-loop structure, and are in keeping with a model proposed for the error-free damage avoidance pathway.

| S-EPMC3300029 | biostudies-literature

Identification of Saccharomyces cerevisiae Genes Whose Deletion Causes Synthetic Effects in Cells with Reduced Levels of the Nuclear Pif1 DNA Helicase.

Project description:The multifunctional Saccharomyces cerevisiae Pif1 DNA helicase affects the maintenance of telomeric, ribosomal, and mitochondrial DNAs, suppresses DNA damage at G-quadruplex motifs, influences the processing of Okazaki fragments, and promotes breakage induced replication. All of these functions require the ATPase/helicase activity of the protein. Owing to Pif1's critical role in the maintenance of mitochondrial DNA, pif1Δ strains quickly generate respiratory deficient cells and hence grow very slowly. This slow growth makes it difficult to carry out genome-wide synthetic genetic analysis in this background. Here, we used a partial loss of function allele of PIF1, pif1-m2, which is mitochondrial proficient but has reduced abundance of nuclear Pif1. Although pif1-m2 is not a null allele, pif1-m2 cells exhibit defects in telomere maintenance, reduced suppression of damage at G-quadruplex motifs and defects in breakage induced replication. We performed a synthetic screen to identify nonessential genes with a synthetic sick or lethal relationship in cells with low abundance of nuclear Pif1. This study identified eleven genes that were synthetic lethal (APM1, ARG80, CDH1, GCR1, GTO3, PRK1, RAD10, SKT5, SOP4, UMP1, and YCK1) and three genes that were synthetic sick (DEF1, YIP4, and HOM3) with pif1-m2.

| S-EPMC4683662 | biostudies-literature

Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1.

Project description:Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins.

| S-EPMC2943577 | biostudies-literature

A 3'-5' exonuclease activity embedded in the helicase core domain of Candida albicans Pif1 helicase.

Project description:3'-5' exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3'-5' exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3'-to-5' polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.

| S-EPMC5316945 | 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