Department of Biology, School of Science, Indiana University Purdue University Indianapolis, Indianapolis, USA.
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, USA.
Curr Genet. 2021 Feb;67(1):85-92. doi: 10.1007/s00294-020-01116-5. Epub 2020 Oct 20.
PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell.
PIF1 家族解旋酶在原核生物和真核生物中具有进化保守性。这些酶通过参与多种 DNA 代谢过程来支持基因组完整性，这些过程可大致分为 DNA 复制、DNA 修复和端粒维持作用。然而，细胞中 PIF1 的活性必须被精细调控，因为在酿酒酵母中 Pif1 的过表达是有毒的，而人源 PIF1（hPIF1）的敲低或过表达则支持癌细胞的生长。这表明 PIF1 家族解旋酶必须在体内受到严格调控，以便将其活性导向需要的位置和时间，并将其活性维持在适当的稳态水平。先前的工作表明，酿酒酵母 Pif1 的 C 端磷酸化调节其端粒维持活性，而我们最近发现 Pif1 还受到赖氨酸乙酰化的调控。在缺乏 Rpd3 赖氨酸去乙酰化酶的细胞中，Pif1 的过表达毒性加剧，但 NuA4 赖氨酸乙酰转移酶亚基 Esa1 的突变则改善了这种毒性。使用重组蛋白，我们发现乙酰化刺激了 Pif1 的 DNA 结合亲和力、ATPase 活性和 DNA 解旋活性。体外实验表明，该解旋酶的三个结构域均为乙酰化的靶标，并且有多项证据表明，乙酰化会导致 Pif1 的 N 端结构域发生构象变化，从而影响这种刺激。目前尚不清楚是什么触发了 Pif1 的赖氨酸乙酰化，以及这种修饰如何影响该酶的许多体内功能，但未来的工作有望阐明该蛋白在细胞内是如何被紧密调控的。
