據(jù)一篇發(fā)表于與10月27日PLoS Biology雜志的研究報(bào)告，美國俄亥俄州立大學(xué)研究人員首次將熒光標(biāo)記物(fluorescent marker)插入聚合酶Dpo4(DNA polymerase IV)的四個結(jié)構(gòu)域和DNA其中一條鏈中,，并利用熒光共振能量轉(zhuǎn)移技術(shù)(fluorescence resonance energy transfer technology)首次實(shí)時(shí)(real-time)觀察到聚合酶結(jié)合到DNA鏈上正確的核苷酸位置并完成DNA復(fù)制的過程,。
在該研究中，根據(jù)聚合酶Dpo4的各個結(jié)構(gòu)域所標(biāo)記的熒光標(biāo)記物顯示,，Dpo4的四個結(jié)構(gòu)域(palm,thumb,finger and little finger)中有三個可緊緊地抓住DNA鏈,，使dNTPs依次與單鏈DNA互補(bǔ)配對完成DNA的復(fù)制，而與前三個結(jié)構(gòu)域相反方向的little finger結(jié)構(gòu)域,，研究人員認(rèn)為可能在DNA復(fù)制過程中,，為錯誤的DNA互補(bǔ)配對留出一定的空間。
研究人員在該研究報(bào)告中,，還將聚合酶結(jié)合到DNA鏈上到復(fù)制出一條完整的DNA鏈的過程明確地分為8個階段,。（生物谷Bioon.com）
相關(guān)研究：
Cell：線粒體DNA復(fù)制酶結(jié)構(gòu)
Nature：研究揭示端粒酶關(guān)鍵部位三維結(jié)構(gòu)
Molecular Cell：解析絲氨酸重組酶DNA復(fù)合體結(jié)構(gòu)
生物谷推薦原始出處：
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.
