Pif1 Activity is Modulated by DNA Sequence and Structure.
Pif1 Activity is Modulated by DNA Sequence and Structure.
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DOI：
10.1021/acs.biochem.1c00614
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发表时间：
2022-01-04
期刊：
影响因子：
2.9
通讯作者：
Bochman ML
中科院分区：
文献类型：
--
作者：
Nickens DG;Bochman ML
编码Pif1解旋酶的基因最初是在酿酒酵母的基因筛选中作为减少线粒体呼吸突变体之间重组的突变体被发现的，随后在影响细胞核端粒长度的基因筛选中被重新发现。现在已知Pif1参与了DNA代谢的许多方面。Pif1的所有已知功能都依赖于与DNA底物的结合，随后是ATP水解，将释放的能量偶联到沿DNA易位以解开双链DNA或其他DNA二级结构。Pif1与高阶DNA结构（如g -四重体DNA）的相互作用以及Pif1装载所需的单链DNA长度已被广泛研究。在这里，为了测试ssDNA长度、序列和结构对Pif1体外生化活性的影响，我们使用了一套基于寡核苷酸的底物来对Pif1 ssDNA结合、atp酶活性和解旋酶活性进行基本表征。使用重组的、未标记的酿酒酵母Pif1，我们发现Pif1优先结合结构化的富含g的ssDNA，但首选的结合底物未能最大限度地刺激atp酶活性。在解旋酶实验中，Pif1浓度低至250 pM时检测到显著的DNA解绕活性。解旋酶实验也表明，Pif1最有效地解绕带有非结构化ssDNA尾部的DNA叉底物。由于Pif1的化学步长已被确定为每次易位或解绕事件1个ATP，这意味着高度结构化的DNA抑制了Pif1中ATP水解与DNA易位和解绕耦合的构象变化。
The gene encoding the Pif1 helicase was first discovered in a Saccharomyces cerevisiae genetic screen as a mutant that reduces recombination between mitochondrial respiratory mutants and was subsequently rediscovered in a screen for genes affecting the telomere length in the nucleus. It is now known that Pif1 is involved in numerous aspects of DNA metabolism. All known functions of Pif1 rely on binding to DNA substrates followed by ATP hydrolysis, coupling the energy released to translocation along DNA to unwind duplex DNA or alternative DNA secondary structures. The interaction of Pif1 with higher-order DNA structures, like G-quadruplex DNA, as well as the length of single-stranded (ss)DNA necessary for Pif1 loading have been widely studied. Here, to test the effects of ssDNA length, sequence, and structure on Pif1’s biochemical activities in vitro, we used a suite of oligonucleotide-based substrates to perform a basic characterization of Pif1 ssDNA binding, ATPase activity, and helicase activity. Using recombinant, untagged S. cerevisiae Pif1, we found that Pif1 preferentially binds to structured G-rich ssDNA, but the preferred binding substrates failed to maximally stimulate ATPase activity. In helicase assays, significant DNA unwinding activity was detected at Pif1 concentrations as low as 250 pM. Helicase assays also demonstrated that Pif1 most efficiently unwinds DNA fork substrates with unstructured ssDNA tails. As the chemical step size of Pif1 has been determined to be 1 ATP per translocation or unwinding event, this implies that the highly structured DNA inhibits conformational changes in Pif1 that couple ATP hydrolysis to DNA translocation and unwinding.
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DOI：
10.1038/nrg3296
发表时间：
2012-11
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
来自 Thermus oshimai 的 SF1B Pif1 的结构和功能研究揭示了二聚化诱导的解旋酶抑制
影响因子：
14.9
作者：
Dai YX;Chen WF;Liu NN;Teng FY;Guo HL;Hou XM;Dou SX;Rety S;Xi XG
通讯作者：
Xi XG
影响因子：
3.8
作者：
Bochman ML;Sabouri N;Zakian VA
通讯作者：
Zakian VA
影响因子：
64.8
作者：
Boulé, JB;Vega, LR;Zakian, VA
通讯作者：
Zakian, VA
影响因子：
2.9
作者：
Barranco-Medina, Sergio;Galletto, Roberto
通讯作者：
Galletto, Roberto