Branched unwinding mechanism of the Pif1 family of DNA helicases

Singh, Saurabh; Soranno, Andrea; Sparks, Melanie; Galletto, Roberto

doi:10.1073/pnas.1915654116

Cited by 17 publications

(21 citation statements)

References 66 publications

(111 reference statements)

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“…While the enzymatic functions of the Pif1 helicase have been extensively characterized, the precise mechanisms by which the activities of this helicase are coordinated to impact a variety of nuclear DNA transactions remain unknown [48][49][50][51][52]. Pif1's cellular abundance is predicted to be low [53], and aberrant Pif1 levels in the cell lead to deleterious effects.…”

Section: Discussionmentioning

confidence: 99%

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Ononye

Sausen

Balakrishnan

et al. 2020

Preprint

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ABSTRACTIn S. cerevisiae, the Pif1 helicase functions to impact both nuclear and mitochondrial DNA replication and repair processes. Pif1 is a 5’-3’ helicase, which preferentially unwinds RNA-DNA hybrids and resolves G-quadruplex structures. Further, regulation of Pif1 by phosphorylation negatively impacts its interaction with telomerase during double strand break repair. Here, we report that in addition to phosphorylation, Pif1 is also modified by lysine acetylation, which influences both its cellular and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 (Esa1) and deacetylase Rpd3 are primarily responsible for dynamically acetylating nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified throughout the protein’s sequence on the N-terminus (K118, K129), helicase domain (K525, K639, K725), and C-terminus (K800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N-terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase activity, while maintaining its substrate preferences. Additionally, both the ATPase and DNA binding activities of Pif1 were stimulated upon acetylation. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose an acetylation-based model for the regulation of Pif1 activities, addressing how this post translational modification can influence its role as a key player in a multitude of DNA transactions vital to the maintenance of genome integrity.

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Section: Discussionmentioning

confidence: 99%

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Ononye

Sausen

Balakrishnan

et al. 2020

Preprint

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“…Briefly, confocal smFRET data were collected on freely diffusing molecules using a MicroTime 200 microscope (PicoQuant) and analyzed using the MatLab based software PAM ( 11 ) to generate FRET histograms from hundreds of events. smFRET-total internal reflection fluorescence (TIRF) experiments were carried out with an objective-type TIRF microscope, essentially as described elsewhere ( 12 , 13 ). Briefly, glass coverslips coated with polyethylene glycol (PEG)-biotin (MicroSurfaces, USA) with additional coverage of 1% Tween-20 to prevent protein sticking to glass coverslips were treated with neutravidin followed by addition of ProT120/478/S525A-Biot.…”

Section: Methodsmentioning

confidence: 99%

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis

2021

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Autoantibodies targeting prothrombin (aPT) can be found in antiphospholipid syndrome (APS) patients. However, their detection has proven difficult to standardize. Here, we developed a new ELISA assay to improve the identification of aPT and compared its performance with currently available anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and autoantibodies targeting prothrombin bound to the plastic plate (aPT-A) assays using a cohort of 27 APS patients at high risk of thrombosis. We generated a novel prothrombin variant, ProTS525A-Biot, carrying an artificial tag at the C-terminus suitable for site-specific biotinylation and added the mutation S525A to improve stability. ProTS525A-Biot was immobilized to neutravidin-coated plates at the desired density and with a defined orientation, i.e., pointing the N-terminal fragment-1 toward the solvent. Antibodies against ProTS525A-Biot (aPT-Bio) were found in 24 out of 27 triple-positive APS patients (88%). When compared to aPS/PT and aPT-A, aPT-Bio showed an excellent linear correlation with aPS/PT (R2 = 0.85) but not with aPT-A (R2 = 0.40). Since aPS/PT but not aPT-A are an emerging biomarker of thrombosis in APS, this method may find utility for detecting pathogenic aPT in APS but also other prothrombotic conditions such as COVID-19.

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“…SmFRET studies have also been performed with forked DNA substrates [ 139 ]. Interestingly, these experiments revealed two classes of unwinding events: repetitive unwinding attempts and full substrate winding [ 139 ].…”

Section: Srs2 and Pif1 As Model Systems For Understanding Sf1a And Sf1b Helicasesmentioning

confidence: 99%

Srs2 and Pif1 as Model Systems for Understanding Sf1a and Sf1b Helicase Structure and Function

Meir

Greene

2021

Genes

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Helicases are enzymes that convert the chemical energy stored in ATP into mechanical work, allowing them to move along and manipulate nucleic acids. The helicase superfamily 1 (Sf1) is one of the largest subgroups of helicases and they are required for a range of cellular activities across all domains of life. Sf1 helicases can be further subdivided into two classes called the Sf1a and Sf1b helicases, which move in opposite directions on nucleic acids. The results of this movement can range from the separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. Here, we describe the characteristics of the Sf1a helicase Srs2 and the Sf1b helicase Pif1, both from the model organism Saccharomyces cerevisiae, focusing on the roles that they play in homologous recombination, a DNA repair pathway that is necessary for maintaining genome integrity.

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Branched unwinding mechanism of the Pif1 family of DNA helicases

Cited by 17 publications

References 66 publications

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

A Novel ELISA Assay for the Detection of Anti-Prothrombin Antibodies in Antiphospholipid Syndrome Patients at High Risk of Thrombosis

Srs2 and Pif1 as Model Systems for Understanding Sf1a and Sf1b Helicase Structure and Function

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