Lysine acetylation regulates the activity of nuclear Pif1

Abstract

In S. cerevisiae, the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA-DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated in vivo. Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1’s cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. Mass spectrometry analysis revealed that Pif1 was modified in several domains 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, ATPase, and DNA binding activities while maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.

Comprehensive Synthetic Genetic Array Analysis of Alleles That Interact with Mutation of the Saccharomyces cerevisiae RecQ Helicases Hrq1 and Sgs1

“…The top five negative and positive interactions for each query strain were confirmed by remaking and reanalyzing the double and triple mutants by hand, followed by spot dilution ( Andis et al 2018 ) and/or growth curve ( Ononye et al 2020 ) assays to compare the growth of the double or triple mutants to their parental strains and wild-type. Examples are shown in Figure S1.…”

The Genetic and Physical Interactomes of theSaccharomyces cerevisiaeHrq1 Helicase

“…The top five positive and negative interactors with hrq1 Δ and hrq1 -K318A from the single-gene deletion and TS arrays were reanalyzed by hand to confirm their phenotypes. Briefly, the SGA query strains MBY639 and MBY644 (Nat R ) were mated to MATa tester strains from the arrays (Kan R ), sporulated, and then analyzed by random spore analysis ( Lichten 2014 ), spot dilution ( Andis et al 2018 ), and/or growth curve ( Ononye et al 2020 ) assays and/or growth curve analyses of Nat R Kan R spore clones compared to the parental single-mutant strains and wild-type.…”

Role and Regulation of Pif1 Family Helicases at the Replication Fork

“…Regulation of Pif1 expression and activity is important, since the DNA helicase is involved in many aspects of replication [ 178 ] and has been linked to breast cancer [ 19 ] and obesity [ 179 ]. Each of the three domains (NTD, helicase, CTD) of ScPif1 contains at least one lysine acetylation site regulated by the acetyltransferase NuA4 and the deacetylase Rpd3 [ 2 ], which may cause a conformational change in the protein [ 2 ]. Interestingly, the overexpression of ScPif1 is toxic [ 4 , 97 ] and the acetylation of the NTD intensifies its toxicity [ 2 ].…”

“…DNA helicases utilize the energy of NTP hydrolysis to translocate on and unwind DNA for processes such as replication and repair. The Pif1 helicase family is part of helicase superfamily 1B [ 1 ], which translocates on single-stranded DNA (ssDNA) and unwinds duplex DNA in the 5′-to-3′ direction [ 2 ]. Pif1 is conserved in all eukaryotes and some bacteria [ 1 ].…”