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chene铁杆木虫 (著名写手)
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[交流]
PLoS Biol：首次实时观察到DNA复制过程中聚合酶的运动
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来源 生命经纬 http://www.bioon.com/biology/Upl ... 009110311180877.jpg 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。 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。 |
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HarveyWang
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小木虫(金币+0.5):给个红包，谢谢回帖交流
小木虫(金币+0.5):给个红包，谢谢回帖交流
楼主的图。  全文链接在此： http://www.plosbiology.org/artic ... ournal.pbio.1000225 可是，大体看了一眼，跟中文的描述不大一样啊，人家是Conformational Dynamics analysis, 可不是”聚合酶的运动“ ！ Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis 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. [ Last edited by HarveyWang on 2009-11-4 at 21:38 ] |
2楼2009-11-04 21:18:53
