Abstract
In Saccharomyces 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. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). 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, whereas 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.
Highlights
At the core of all cellular transactions, such as replication, repair, and transcription, is the need for biological machines to gain access to the genetic information stored within the DNA duplex [1]
Because the sequence coding for Pif1 is fairly rich in lysine residues (;10% of the whole sequence), an amino acid that serves as a good target for many post-translational modifications (PTMs), we were interested in investigating the acetylation dynamics of Pif1
It has previously been reported that the overexpression of Pif1 in S. cerevisiae is toxic to cell growth [35,36,37]
Summary
At the core of all cellular transactions, such as replication, repair, and transcription, is the need for biological machines to gain access to the genetic information stored within the DNA duplex [1]. 1% of the genes in eukaryotic genomes code for helicases, and to date, over 100 RNA and DNA helicases have been discovered [2, 3] These motor proteins function by coupling ATP hydrolysis to mechanical movement to break the hydrogen bonds between complementary base pairs in dsDNA [2]. Following its initial discovery in yeast mitochondria over 20 years ago, Pif has been shown to localize to the nucleus, where it participates in a myriad of DNA transactions It functions in Okazaki fragment maturation, wherein it lengthens the flap ahead of DNA polymerase d, allowing RPA to bind to the displaced flap [11]. The results of our cellular assays and in vitro studies indicate that lysine acetylation serves as an important regulator of Pif activity within the cell
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