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首次实时观察到DNA复制过程中聚合酶的运动
分类:国际医学技术 来源:本站编辑部 作者:med00 职称:
摘要:
研究人员在该研究报告中,还将聚合酶结合到DNA链上到复制出一条完整的DNA链的过程明确地分为8个阶段。

注:聚合酶与DNA结合示意图。金色为DNA序列,图中蓝色、红色、绿色、紫色分别代表结构域finger、palm、thumb、LF

据一篇发表于与10月27日PLoS Biology杂志的研究报告,美国俄亥俄州立大学研究人员首次将荧光标记物(fluorescent marker)插入聚合酶Dpo4(DNA polymerase IV)的四个结构域和DNA其中一条链中,并利用荧光共振能量转移技术(fluorescence resonance energy transfer technology)首次实时(real-time)观察到聚合酶结合到DNA链上正确的核苷酸位置并完成DNA复制的过程。

在该研究中,根据聚合酶Dpo4的各个结构域所标记的荧光标记物显示,Dpo4的四个结构域(palm,thumb,finger and little finger)中有三个可紧紧地抓住DNA链,使dNTPs依次与单链DNA互补配对完成DNA的复制,而与前三个结构域相反方向的little finger结构域,研究人员认为可能在DNA复制过程中,为错误的DNA互补配对留出一定的空间。

相关研究:

Cell:线粒体DNA复制酶结构

Nature:研究揭示端粒酶关键部位三维结构

Molecular Cell:解析丝氨酸重组酶DNA复合体结构

原始出处:

PLoS Biol 7(10): e1000225. doi:10.1371/journal.pbio.1000225

Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis

Cuiling Xu1, Brian A. Maxwell2, Jessica A. Brown1,3, Likui Zhang1, Zucai Suo1,2,3,4,5*

1 Department of Biochemistry, The Ohio State University, Columbus, Ohio, United States of America, 2 Ohio State Biophysics Program, The Ohio State University, Columbus, Ohio, United States of America, 3 Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, United States of America, 4 Molecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio, United States of America, 5 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, United States of America

Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.

 

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浏览次数:3589次   发布日期:2009-12-24

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