用荧光显微示踪方法研究RecA在DNA同源识别过程中的工作机理

庞哲; 王爽; 李辉; 徐春华; 李明

doi:10.7498/aps.61.218701

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摘要

RecA是原核生物体内参与DNA同源识别过程的一种关键蛋白, 长期以来一直是同源重组相关课题的重要研究对象. 通过荧光显微示踪方法, 发现在同源识别过程中 RecA与单链DNA形成的核蛋白丝与模板DNA的结合是短时(τ=0.2 s)和短程(l=1.05 μm)的, 结合后搜寻模板DNA上的同源位点的过程可分为布朗运动和定向运动两种模式. 结合时核蛋白丝并不是缠绕在模板DNA上, 而是以一种更弱的方式结合在模板DNA外侧进行位点搜寻. 如果在该过程中没有找到同源位点, 核蛋白丝就会脱离模板DNA, 并寻找下一次与模板DNA结合的机会, 重复以上过程.

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作者及机构信息

1.
北京凝聚态物理国家实验室, 中国科学院物理研究所软物质物理重点实验室, 北京 100190

基金项目: 国家自然科学基金(批准号: 10927402, 11004234)资助的课题.

参考文献

[1]	Muskavitch K M T, Linn S 1981 Enzymes 14 233
[2]	Maser R S, Monsen K J, Nelms B E, Petrini J H 1997 Mol. Cell. Biol. 17 6087
[3]	Benson F E, Stasiak A, West S C 1994 EMBO. J. 13 5764
[4]	New J H, Sugiyama T, Zaitseva E, Kowalczykowski S C 1998 Nature 391 407
[5]	Constantinou A, Davies A A, West S C 2001 Cell 104 259
[6]	Heyer W, Ehmsen K T, Solinger J A 2003 Trends Biochem. Sci. 28 548
[7]	Shan Q, Cox M M 1997 J. Biol. Chem. 272 11063
[8]	Heijden T V D, Modesti M, Hage S, Kanaar R, Wyman C, Dekker C 2008 Cell 30 530
[9]	Joo C, Mckinney S A, Nakamura M, Rasnik I, Myong S, Ha T 2006 Cell 126 515
[10]	Forget A L, Kowalczykowski S C 2012 Nature 482 423
[11]	Story R M, Weber I T, Steitz T A 1992 Nature 355 318
[12]	Tafvizi A, Huang F, Fersht A R, Mirny L A, Oijen A M V 2011 Proc. Natl. Acad. Sci. 108 563
[13]	Gorman J, Chowdhury A, Surtees J A, Shimada J, Reichman D R, Alani E, Greene E C 2007 Cell 28 359
[14]	Saxton M J, Jacobson K 1997 Annu. Rev. Biophys. Biomol. Struct. 26 373
[15]	Graneli A, Yeykal C C, Robertson R B, Greene E C 2006 Proc. Natl. Acad. Sci. 103 1221
[16]	Li H, Duan Z W, Dou S X, Wang P Y 2012 Acta Phys. Sin. 61 068701 (in Chinese) [李辉, 段兆文, 窦硕星, 王鹏业 2012 物理学报 61 068701]
[17]	Bruinsma R F 2002 Physica A 313 211
[18]	Nishinaka T, Shinohara A, Ito Y, Yokoyama S, Shibata T 1998 Proc. Natl. Acad. Sci. 95 11071
[19]	Bagchi B, Blainey P C, Xie X S 2008 J. Phys. Chem. B 112 6282
[20]	Blainey P C, Luo G B, Kou S C, Mangel W F, Verdine G L, Bagchi B, Xie X S 2009 Nature Struct. Mol. Biol. 16 1224
[21]	PHsieh P, Camerini-Otero C S, Camerini-Otero R D 1992 Proc. Natl. Acad. Sci. 89 6492
[22]	Rosselli W, Stasiak A 1990 J. Mol. Biol. 216 35
[23]	Stasiak A, Capua E D 1982 Nature 299 185

施引文献

[1]	Muskavitch K M T, Linn S 1981 Enzymes 14 233
[2]	Maser R S, Monsen K J, Nelms B E, Petrini J H 1997 Mol. Cell. Biol. 17 6087
[3]	Benson F E, Stasiak A, West S C 1994 EMBO. J. 13 5764
[4]	New J H, Sugiyama T, Zaitseva E, Kowalczykowski S C 1998 Nature 391 407
[5]	Constantinou A, Davies A A, West S C 2001 Cell 104 259
[6]	Heyer W, Ehmsen K T, Solinger J A 2003 Trends Biochem. Sci. 28 548
[7]	Shan Q, Cox M M 1997 J. Biol. Chem. 272 11063
[8]	Heijden T V D, Modesti M, Hage S, Kanaar R, Wyman C, Dekker C 2008 Cell 30 530
[9]	Joo C, Mckinney S A, Nakamura M, Rasnik I, Myong S, Ha T 2006 Cell 126 515
[10]	Forget A L, Kowalczykowski S C 2012 Nature 482 423
[11]	Story R M, Weber I T, Steitz T A 1992 Nature 355 318
[12]	Tafvizi A, Huang F, Fersht A R, Mirny L A, Oijen A M V 2011 Proc. Natl. Acad. Sci. 108 563
[13]	Gorman J, Chowdhury A, Surtees J A, Shimada J, Reichman D R, Alani E, Greene E C 2007 Cell 28 359
[14]	Saxton M J, Jacobson K 1997 Annu. Rev. Biophys. Biomol. Struct. 26 373
[15]	Graneli A, Yeykal C C, Robertson R B, Greene E C 2006 Proc. Natl. Acad. Sci. 103 1221
[16]	Li H, Duan Z W, Dou S X, Wang P Y 2012 Acta Phys. Sin. 61 068701 (in Chinese) [李辉, 段兆文, 窦硕星, 王鹏业 2012 物理学报 61 068701]
[17]	Bruinsma R F 2002 Physica A 313 211
[18]	Nishinaka T, Shinohara A, Ito Y, Yokoyama S, Shibata T 1998 Proc. Natl. Acad. Sci. 95 11071
[19]	Bagchi B, Blainey P C, Xie X S 2008 J. Phys. Chem. B 112 6282
[20]	Blainey P C, Luo G B, Kou S C, Mangel W F, Verdine G L, Bagchi B, Xie X S 2009 Nature Struct. Mol. Biol. 16 1224
[21]	PHsieh P, Camerini-Otero C S, Camerini-Otero R D 1992 Proc. Natl. Acad. Sci. 89 6492
[22]	Rosselli W, Stasiak A 1990 J. Mol. Biol. 216 35
[23]	Stasiak A, Capua E D 1982 Nature 299 185

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用荧光显微示踪方法研究RecA在DNA同源识别过程中的工作机理

1. 北京凝聚态物理国家实验室, 中国科学院物理研究所软物质物理重点实验室, 北京 100190

基金项目:

国家自然科学基金(批准号: 10927402, 11004234)资助的课题.

收稿日期: 2012-04-15

修回日期: 2012-05-23

刊出日期: 2012-11-05

摘要: RecA是原核生物体内参与DNA同源识别过程的一种关键蛋白, 长期以来一直是同源重组相关课题的重要研究对象. 通过荧光显微示踪方法, 发现在同源识别过程中 RecA与单链DNA形成的核蛋白丝与模板DNA的结合是短时(τ=0.2 s)和短程(l=1.05 μm)的, 结合后搜寻模板DNA上的同源位点的过程可分为布朗运动和定向运动两种模式. 结合时核蛋白丝并不是缠绕在模板DNA上, 而是以一种更弱的方式结合在模板DNA外侧进行位点搜寻. 如果在该过程中没有找到同源位点, 核蛋白丝就会脱离模板DNA, 并寻找下一次与模板DNA结合的机会, 重复以上过程.

关键词:

A study on the mechanism of RecA in homologous recognition by using single molecule fluorescence tracking

1.
Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China

Fund Project:

Project supported by the National Natural Science Foundation of China (Grant Nos. 10927402, 11004234).

Received Date: 15 April 2012

Accepted Date: 23 May 2012

Published Online: 05 November 2012

Abstract: RecA plays an important role in homologous recognition in prokaryotes, and it has become a hot point in homologous recognition related research since its discovery. We establish an assay by combining total internal reflection fluorescence and flow stretching to visualize in real time the motion of single RecA-ssDNA filaments which are tagged with fluorescent beads. This enables us to study the interaction of RecA-ssDNA filaments with their templates in the homologous recognition process. It is found that the searching and binding is a short-time (τ=0.2 s) and short-distance (l=1.05 μm) process. Two distinguished motion modes for the RecA-ssDNA filament are observed, a Brownian motion and a directed motion. The observations suggest a model that a RecA-ssDNA filament just interacts weakly with the template DNA before it binds firmly to the template DNA. If no homologous site is found in a searching process, the filament drops off the template and repeats the searching process again until it finally finds its target.

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