Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA
Mechanism of ATP-dependent translocation of E. coli UvrD monomers along single-stranded DNA
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DOI：
10.1016/j.jmb.2004.10.005
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发表时间：
2004-12-10
影响因子：
5.6
通讯作者：
Lohman, TM
中科院分区：
文献类型：
--
作者：
Fischer, CJ;Maluf, NK;Lohman, TM
大肠杆菌UvrD蛋白是一种3'到5' SF 1 DNA解旋酶，参与DNA的甲基指导错配修复和核苷酸切除修复。利用停流法，我们已经研究了UvrD单体的动力学机制，沿着单链DNA（ssDNA）在体外易位，通过监测蛋白质到达5的ssDNA的瞬时动力学。通过蛋白质对连接到一系列长度从16到124 nt变化的寡脱氧胸苷酸的5 ′-末端的荧光团（Cy 3或荧光素）的荧光强度的影响来监测到达5 ′-末端。我们发现UvrD单体能够沿着沿着ssDNA以偏置的3'至5'方向性进行ATP依赖性易位。全球非线性最小二乘分析的完整动力学时间过程中存在的蛋白质陷阱，以防止重新绑定的游离蛋白质的DNA使用所附文件中描述的方法，使我们能够获得定量估计的动力学参数易位。我们发现，UvrD单体在不连续的步骤，平均动力学步长，m=3.68（+/-0.03）nt步骤（-1），易位速率常数，k（t）=51.3（+/-0.6）步s（-1），（宏观易位率，δ（t）= 189.0（+/-0.7）nt s（-1）），其中持续合成能力对应于解离前2400（+/-600）nt的平均易位距离（10 mM Tris-HCl（pH 8.3），20 mM NaCl，20%（v/v）甘油，25 ℃）。然而，尽管UvrD单体能够沿着ssDNA快速且有效地易位，但其在体外甚至不能解旋18 bp双链体。DNA解旋酶的体外活性需要一个UvrD二聚体，在相同的溶液条件下，UvrD二聚体解旋DNA的动力学步长为4-5 bp step（-1），但解旋速率慢三倍，为68（+/-9）bp s（-1），这表明DNA解旋活性需要的不仅仅是简单地沿ss-DNA定向沿着的能力。(C)2004 Elsevier Ltd.保留所有权利。
Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5 of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global nonlinear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, k(t)=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mk(t)= 189.0(+/-0.7)nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600)nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degreesC). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but a similar tothreefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA. (C) 2004 Elsevier Ltd. All rights reserved.