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

Nickens, David G.; Sausen, Christopher W.; Bochman, Matthew L.

doi:10.3390/genes10060411

Cited by 21 publications

(38 citation statements)

References 39 publications

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“…The results of these experiments are shown in Figure 1. Here, we recapitulated the toxicity of Pif1 overexpression reported by others [35,36], finding an approximately 50-60% reduction in wild-type growth upon overexpression of the helicase ( Figure 1A).…”

Section: Cellular Acetylation Status Modulates Pif1 Overexpression Tosupporting

confidence: 84%

“…To understand the impact of global cellular acetylation on the function of Pif1, we initially sought to alter the acetylation dynamics in the cell by creating either KAT or KDAC mutant strains. It has previously been reported that the overexpression of Pif1 in S. cerevisiae is toxic to cell growth [35,36]. Therefore, we used this overexpression toxicity phenomenon to develop a phenotypic assay to determine the impact of cellular acetylation.…”

Section: Cellular Acetylation Status Modulates Pif1 Overexpression Tomentioning

confidence: 99%

“…A wealth of research has improved our understanding of the structural motifs and related functions of the helicase domain, but in comparison, little is known about the N-and C-terminal domains of Pif1. Currently, these regions are postulated to be modular accessory domains that may serve as regulatory regions [9,19,20]. We hypothesize that the functional significance of these domains may help to maintain specific folds that are necessary for protein function and establish points for post translational modifications (PTMs) [21].…”

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confidence: 99%

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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: Cellular Acetylation Status Modulates Pif1 Overexpression Tosupporting

confidence: 84%

Section: Cellular Acetylation Status Modulates Pif1 Overexpression Tomentioning

confidence: 99%

mentioning

confidence: 99%

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Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Ononye

Sausen

Balakrishnan

et al. 2020

Preprint

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“…Whereas Lc and Kc did not differ between loci located more than 33kb away from the centromere, the coefficients of diffusion (D) were similar for the 12kb and 33kb centromere proximal loci but significantly increased for loci more than 50kb away from the centromere ( Figure 1G). As previously described, all the loci analysed showed sub-diffusive motion, with mean values for alpha ranging from alpha 12kb =0.36 to alpha 116kb = 0.51 and a large cell-to-cell variation 19,26,34,35,37,38 . As for D, alpha values were low and indistinguishable for the two centromere proximal loci at 12 and 33 kb and significantly higher for loci located beyond 50 kb ( Figure 1H).…”

Section: Differential Centromere and Telomere Mobilitysupporting

confidence: 64%

Telomeric chromosome ends are highly mobile and behave like free double-strand DNA breaks

Toulouze

Amitai

Shukron

et al. 2019

Preprint

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wordsChromosome organization and dynamics are critical for DNA transactions, including gene expression, replication, and DNA repair. In yeast, the chromosomes are anchored through their centromeres to the spindle pole body, and their telomeres are grouped into clusters at the nuclear periphery, constraining chromosome mobility. Here, we have used experimental and computational approaches to study the effects of chromosome-nuclear envelope (NE) attachments on the dynamics of S. cerevisiae chromosomes. We found that although centromere proximal loci were, as predicted, more dynamically constrained than distal loci, telomeres were highly mobile, even when positioned at the nuclear periphery. Polymer modeling indicated that polymer ends are intrinsically more mobile than internal sites. We tested this model by measuring the mobility of a double strand break (DSB) end within a chromosome arm. Upon separation of the DSB ends, their mobility significantly increased.Altogether, our results reveal that telomeres behave as highly mobile polymer ends, despite interactions with the nuclear membrane. Main Text: IntroductionGenome organization is key in controlling genomic functions such as gene expression, DNA damage repair and replication 1-4 . The arrangement of the genome in the nucleus has been described from analysis of loci in population of cells, revealing preferential positions relative to fixed nuclear structures, such as the nuclear envelope (NE) and nucleolus. Chromatin contacts within and between chromosomes and with nuclear structures determine how the genome is folded, which has consequences for its function [5][6][7][8][9] . However how these positions change dynamically in single cells is not fully understood.The 16 chromosomes in the budding yeast S. cerevisiae are bound to the NE through the several interactions. Their point centromeres bind the kinetochores attached to the spindle pole body (SPB), which is embedded into the NE along the whole cell cycle 10-12 . During interphase yeast chromosomes are also tethered to the NE via their telomeres through several pathways involving YKU, Sir4, Esc1, Csm4 Mps3 and the nuclear pore complex (NPC) 13-21 . In addition, Telomeres cluster into 4-6 perinuclear foci at the NE, which favours transcriptional repression mediated by the SIR complex and limits SIR access to other sites of the genome 22-24 . Telomere tethering in S phase contributes also to telomerase control and suppresses recombination among telomere repeats 20,25 . Several intrachromosomal loci, mostly corresponding to either highly transcribed or inducible genes when activated, interact with the NPC imbedded in the NE 26-32 , an interaction that may provide additional layer of regulation and promote optimal gene expression 27,28,33 .In all organisms genome arrangement varies from cell to cell, and over time, within single cells, due to chromatin motion (visualized using fluorescent repressor operator system [FROS] tagging of single loci and followed at milliseconds time resolution). Chromatin exhibits a...

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“…Genome integrity is also highlighted by CBC2, which encodes an RNA binding and processing factor involved in telomere maintenance (LEE-SOETY et al 2012). Hrq1 is known to regulate telomerase activity at both DSBs and telomeres (BOCHMAN et al 2014;NICKENS et al 2018;NICKENS et al 2019). Mutation of the gene encoding the Vps41 vacuolar membrane protein (NAKAMURA et al 1997) also negatively interacted with hrq1-K318A.…”

Section: Hrq1-k318a Interactionsmentioning

confidence: 99%

Comprehensive synthetic genetic array analysis of alleles that interact with mutation of theSaccharomyces cerevisiaeRecQ helicases Hrq1 and Sgs1

Sanders

Nguyen

Rogers

et al. 2020

Preprint

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Most eukaryotic genomes encode multiple RecQ family helicases, including five such enzymes in humans. For many years, the yeast Saccharomyces cerevisiae was considered unusual in that it only contained a single RecQ helicase, named Sgs1. However, it has recently been discovered that a second RecQ helicase, called Hrq1, resides in yeast. Both Hrq1 and Sgs1 are involved in genome integrity, functioning in processes such as DNA inter-strand crosslink repair, double-strand break repair, and telomere maintenance. However, it is unknown if these enzymes interact at a genetic, physical, or functional level as demonstrated for their human homologs. Thus, we performed synthetic genetic array (SGA) analyses of hrq1Δ and sgs1Δ mutants. As inactive alleles of helicases can demonstrate dominant phenotypes, we also performed SGA analyses on the hrq1-K318A and sgs1-K706A ATPase/helicase-null mutants, as well as all combinations of deletion and inactive double mutants. We crossed these eight query strains (hrq1Δ, sgs1Δ, hrq1-K318A, sgs1-K706A, hrq1Δ sgs1Δ, hrq1Δ sgs1-K706A, hrq1-K318A sgs1Δ, and hrq1-K318A sgsl-K706A) to the S. cerevisiae single gene deletion and temperature-sensitive allele collections to generate double and triple mutants and scored them for synthetic positive and negative genetic effects based on colony growth. These screens identified hundreds of synthetic interactions, supporting the known roles of Hrq1 and Sgs1 in DNA repair, as well as suggesting novel connections to rRNA processing, mitochondrial DNA maintenance, transcription, and lagging strand synthesis during DNA replication.

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

Cited by 21 publications

References 39 publications

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Nuclear Pif1 is Post Translationally Modified and Regulated by Lysine Acetylation

Telomeric chromosome ends are highly mobile and behave like free double-strand DNA breaks

Comprehensive synthetic genetic array analysis of alleles that interact with mutation of theSaccharomyces cerevisiaeRecQ helicases Hrq1 and Sgs1

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