Lysine acetylation regulates the activity of nuclear Pif1
Lysine acetylation regulates the activity of nuclear Pif1
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DOI：
10.1074/jbc.ra120.015164
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发表时间：
2020-11-13
影响因子：
4.8
通讯作者：
Bochman, Matthew L.
中科院分区：
文献类型：
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作者：
Ononye, Onyekachi E.;Sausen, Christopher W.;Bochman, Matthew L.
在酿酒酵母中，Pif1解旋酶在细胞核和线粒体DNA的复制和修复过程中都起作用，优先解绕RNA:DNA杂交体和分解g -四重体结构。我们试图确定Pif1的各种活性是如何在体内被调节的。在这里，我们报道了核Pif1的赖氨酸乙酰化，并证明它影响Pif1的细胞作用和核心生化活性。通过Pif1过表达毒性实验，我们确定乙酰转移酶NuA4和去乙酰化酶Rpd3是核Pif1动态乙酰化的主要原因。质谱分析显示，Pif1在蛋白序列的N端（Lys-118和Lys-129）、解旋酶结构域（Lys-525、Lys-639和Lys-725）和C端（Lys-800）的多个区域被修饰。Pif1的乙酰化加剧了其过表达毒性表型，在删除其N端后减轻了这种毒性表型。生化分析表明，Pif1的乙酰化刺激了它的解旋酶、atp酶和dna结合活性，同时保持了它的底物偏好。有限的蛋白水解实验表明，Pif1的乙酰化引起构象变化，这可能是其酶性质改变的原因。我们认为乙酰化参与了Pif1活性的调节，影响了对维持基因组完整性至关重要的大量DNA交易。
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.