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大肠杆菌单链结合蛋白（E.coli SSB）具有与单链DNA（single-stranded DNA，ssDNA）结合的性质，起着保护ssDNA及引导相关蛋白质反应的重要作用，然而，它与ssDNA的结合过程及其细节尚未得到确定的研究结果.本文采用单分子磁镊对E.coli SSB/ssDNA复合体进行拉力测试，并采用单分子化学反应动力学方法对测试结果进行分析.研究发现：E.coli SSB在ssDNA上的结合过程分为两个不同的阶段，一个是在临界力下快速的结合缠绕阶段，一个是随着力的进一步减小逐步缠绕的阶段.从中得到了E.coli SSB与ssDNA的化学反应系数，并得到了相应的自由能参数.采用自由能校准的方法，得到了SSB与ssDNA结合的完整自由能曲线，从而揭示了E.coli SSB与ssDNA的结合特征.本文的分析方法也可以应用于相似的化学反应的研究中.Single-stranded DNA binding proteins (SSBs) widely exist in different kinds of creatures. It can bind single-stranded DNA (ssDNA) with high affinity. The binding is sequence independent. SSB can also interact with different kinds of proteins, and thus leading them to work at the special sites. It plays an essential role in cell metabolism. E.coli SSB is a representative of SSB among all kinds of SSBs, it is a homotetramer consisting of four 18.9 kD subunits, the homotetramer is stable under low concentration. E.coli SSB has different binding modes under different salt concentrations (for example NaCl). When NaCl concentration is higher than 200 mM, E.coli SSB can bind 65 nt ssDNA, when NaCl concentration is lower than 20 mM, it can bind 35 nt ssDNA, and when the NaCl concentration is between 20 mM and 200 mM, it can bind 56 nt ssDNA. The characteristics of E.coli SSB are so attractive that a large number of researches have been done to distinguish its binding process. Earlier researchers tried to use stop flow technology to study the interaction between SSB and ssDNA in bulk. However, the high affinity between SSB and ssDNA makes this interaction too rapid to be observed at all, and the dissociate interaction even could not be measured. Single molecule technology which combines with low and accurate force offers researchers another way to achieve this goal. Some researchers observed the unwrapping phenomenon in an optical tweezers pulling experiment. However, they did not find the detailed process of binding or dissociation. In our work, we use a magnetic tweezer to pull the SSB/ssDNA complex and find a special phenomenon like double-state jump. Using the single molecule dynamics to analyse the data, we find that this phenomenon is the combination and dissociation between SSB and ssDNA. After comparing the pulling curve of ssDNA only and SSB/ssDNA complex, we find that the SSB binding process consists of two stages, one is rapid combination/dissociation under the action of a critical force; the other is continuous wrapping following the reduced force. According to Bell formula and SSB/ssDNA complex binding model, we obtain the interaction rate and free energy parameters under 0 pN, and we calibrate the free energy to obtain its continuous wrapping part, so we can obtain the whole free energy landscape and understand the binding process. Our analysis way is also applicable to the case of similar interactions to obtain their interaction details and free energy characteristics.
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Keywords:
- single-molecule dynamics /
- single-stranded DNA binding proteins /
- binding process /
- free energy
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[1] Chase J W, Williams K R 1986 Annu. Rev. Biochem. 55 103
[2] Meyer R R, Laine P S 1990 Microbiol. Rev. 54 342
[3] Yuzhakov A, Kelman Z, O'Donnell M 1999 Cell 96 153
[4] Sun W, Godson G N 1998 J. Mol. Biol. 276 689
[5] Lohman T M, Ferrari M E 1994 Annu. Rev. Biochem. 63 527
[6] Shereda R D, Kozlov A G, Lohman T M, Cox M M, Keck J L 2008 Crit. Rev. Biochem. Mol. Biol. 43 289
[7] Fu H X, Le S M, Chen H, Muniyappa K, Yan J 2013 Nucleic Acids Res. 41 924
[8] Bell J C, Plank J L, Dombrowski C C, Kowalczykowski S C 2012 Nature 491 274
[9] Waldman V M, Weiland E, Kozlov A G, Lohman T M 2016 Nucleic Acids Res. 44 4317
[10] Raghunathan S, Ricard C S, Lohman T M, Waksman G 1997 Proc. Natl. Acad. Sci. USA 94 6652
[11] Bujalowski W, Lohman T M 1986 Biochemistry 25 7799
[12] Kozlov A G, Cox M M, Lohman T M 2010 J. Biol. Chem. 285 17246
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[20] Wang S, Qin W, Li J H, Lu Y, Lu K Y, Nong D G, Dou S X, Xu C H, Xi X G, Li M 2015 Nucleic Acids Res. 43 3736
[21] Wang S, Zheng H Z, Zhao Z Y, Lu Y, Xu C H 2013 Acta Phys. Sin. 62 168703 (in Chinese)[王爽, 郑海子, 赵振业, 陆越, 徐春华 2013 物理学报 62 168703]
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[23] Xie P, Dou S X, Wang P Y 2004 Chin. Phys. 13 1569
[24] Zhao Z Y, Xu C H, Shi J, Li J H, Ma J B, Jia Q, Ma D F, Li M, Lu Y 2017 Chin. Phys. B 26 088701
[25] Saleh O A, McIntosh D B, Pincus P, Ribeck N 2009 Phys. Rev. Lett. 102 068301
[26] Kramers H A 1940 Physica 7 284
[27] Dudko O K, Hummer G, Szabo A 2008 Proc. Natl. Acad. Sci. USA 105 15755
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[29] Pope L H, Bennink M L, van Leijenhorst-Groener K A, Nikova D, Greve J, Marko J F 2005 Biophys. J. 88 3572
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[32] Bujalowski W, Lohman T M 1989 J. Mol. Biol. 207 269
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