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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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Keywords:
- RecA /
- homologous recognition /
- single molecule tracking /
- flow stretching
[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 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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[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 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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