解旋酶Sen1行走机制的研究

Mechanism of Sen1 translocation

转录终止是调节基因转录过程的重要步骤,关系到基因表达调控过程的正常进行和基因组的稳定性.酵母体系中,Sen1是一种解旋酶,通过水解腺苷三磷酸(adenosine triphosphate,ATP)获得能量来行使在核酸底物上的一维运动,进而实现转录终止功能.然而,Sen1的这种运动机制与转录终止功能的关联,目前尚未清晰的表征.本文应用凝胶电泳迁移方法表征Sen1解旋双链DNA和结合单链DNA的能力,并且发现单个Sen1分子能够结合≤24 nt(nucleotide,nt)的单链DNA;进一步,在提供ATP的条件下,应用单分子荧光共振能量转移技术表征了Sen1在单链DNA上的行走功能,其行走速率随ATP浓度变化而变化,符合经典米氏动力学模型;考虑单链DNA底物的长度,可以估算出Sen1的行走速率约为70 nt/s.这些研究结果定量表征了Sen1的行走速率,为理解真核转录终止机制提供了分子马达运动方面的实验数据.

Transcription termination is a critical step for gene regulation and genome integrity among all kingdoms of life.In Saccharomyces cerevisiae,one of the major termination pathways is accomplished by Sen1 helicase,a homolog to human Senataxin(SETX),defection of which raises the diseases for the central nervus system of human.Although it has been proposed that Sen1 translocates along nucleic acids by consuming adenosine triphosphates(ATPs)during termination,the mechanism for this translocation activity of Sen1 has not been well understood.In this work,our aim is to investigate the mechanism of Sen1 translocation by measuring the interactions between Sen1 and different types of nucleic acids by polyacrylamide gel electrophoresis(PAGE)assay or single-molecule Fӧrster resonance energy transfer(FRET)assay.We firstly observe the unwinding activity of Sen1 on a tailed duplex DNA in the presence of 1 mM ATP via PAGE assay,where the translocation activity of Sen1 is involved.As the binding activity is crucial for translocation,then we examine the binding affinity of Sen1 to the single-stranded DNA via PAGE assay,revealing a stable binding of Sen1 with an occupied length of nucleic acids of less than 24 nt.In the presence of 1μM ATP,we observe that Sen1 dynamically binds to and dissociates from the tailed duplex DNA in the single-molecule FRET assay.By titrating ATP concentrations from 1-500μM,we observe a gradual decrease in the mean durations of Sen1 binding,suggesting an ATP-dependent binding affinity of Sen1 to single-stranded DNA.We then fit these mean durations to the classical Michaelis-Menten model and obtain a minimum binding duration of(0.18±0.01)s at saturating ATP concentrations and Km of(13.1±0.1)μM for the ATP-dependent binding of Sen1.This result is consistent with that from a translocation activity of Sen1.Taking into account the translocation length of the half of the single-stranded tail,i.e.13 nt,a mean rate of 70 nt/s is estimated.Reversing the translocation direction,we observe an increase in the duration of Sen1 binding to the single-stranded tail,which suggests an impediment of DNA duplex in front of Sen1 translocation or the possible duplex DNA unwinding activity of Sen1.Our quantitative measurements on Sen1 translocation are helpful in deepening our understanding of the mechanism of eukaryotic transcription termination by Sen1.