Atomic Force Microscopic Study of Low Temperature Induced

Jan 1, 2008 - The assembly and disassembly of RecA−DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are ...
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J. Phys. Chem. B 2008, 112, 1022-1027

Atomic Force Microscopic Study of Low Temperature Induced Disassembly of RecA-dsDNA Filaments Cunlan Guo, Yonghai Song, Li Wang, Lanlan Sun, Yujing Sun, Chongyang Peng, Zhelin Liu, Tao Yang, and Zhuang Li* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China ReceiVed: September 9, 2007; In Final Form: October 22, 2007

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments. In addition, when the formed filaments were kept at -20 °C for 20 h with 1,4-dithiothreitol (DTT), the integrated filaments disassembled. It was similar to the case under the same condition without anything added. However, when glycerol was used as a substitute for DTT, there was no obvious disassembly at the same condition. Oppositely, when the formed filaments were kept at 4 °C for 20 h, the disassembly with additional DTT was not as obvious as the case at -20 °C for 20 h, whereas the case with additional glycerol disassembled. The experiments indicated the effect of cold denaturation on the interaction of DNA and RecA. Meanwhile, the study of these phenomena can supply guidelines for the property and stability of RecA as well as the relevant roles of influencing factors to RecA and DNA in further theoretical studies.

Introduction The RecA of Escherichia coli and its homologues as recombinase generally exist in nature and play essential roles in many biological functions, especially in the coupled “3R” processes of replication, recombination, and repair. No matter any functions of RecA, it is a tacit assumption that all need the formation of active nucleoprotein filaments first.1,2 In the first phase of the homologous recombination, RecA can assemble on single-stranded DNA (ssDNA) or doublestranded DNA (dsDNA) containing nicks or single-stranded gaps to form an integrated nucleoprotein filament with ATP or its nonhydrolyzed analogue ATPγS.2-8 The interaction of RecA with ssDNA and dsDNA is similar in many aspects. Many studies indicate that RecA reacts with both ssDNA and dsDNA in the presence of ATP during strand exchange reactions.9 However, the reaction of RecA and ssDNA is more complicated as ssDNA-binding (SSB) protein is often needed to remove the secondary structures of ssDNA in order to promote this reaction,10,11 so dsDNA is usually used as a model to research the property and dynamics of interaction between RecA and DNA. The assembly and disassembly of the active nucleoprotein filaments are critical to the DNA strand exchange reactions of homologous recombination, and the dissociated RecA protein can cycle to perform its functions in living cells. Up to now, a * To whom correspondence should be addressed at State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China. Phone: +86 431 85262057. Fax: +86 431 85262057. E-mail: [email protected].

lot of studies have been focused on the assembly and disassembly of the RecA nucleoprotein filament including reaction rate, reaction phase, and influencing factors. A large number of methods have been used in these studies such as ATPase assay,12,13 electron microcopy,14 spectrophotometric assay,15 circular dichroism spectroscopy,16 light scattering,17 fluorescence microscopy,18-21 laser tweezers,9,22 and magnetic tweezers.23,24 For example, an early electron microscopic study indicated that the binding of RecA to dsDNA partially unwound the DNA and elongated the DNA by 50%. A slow nucleation and fast growth mechanism has been proposed for the assembly of RecA and by Pugh and Cox based on spectrophotometric monitoring of ATP hydrolysis, DNase resistance, and light scattering studies.25 Atomic force microscopy (AFM) is considered to be a useful tool in biochemical studies. The samples of AFM imaging can be easily prepared in either an ambient or liquid environment without staining or fixing. Furthermore, because of its highresolution visualization, the size of individual molecules can be accurately determined and also the refined structure and the slight change of protein-mediated interactions can be directly observed.26 AFM has also been employed to study the RecA nucleoprotein filaments due to its intrinsic advantages over other methods.27-30 Here we report an AFM study of low temperature induced disassembly of RecA-dsDNA-APTγS filaments. In spite of intensive study, the detailed mechanism and refined influencing factors for assembly and disassembly of RecA nucleoprotein filament are not completely understood, with the reason that the processes can be influenced by various aspects such as multiple reaction steps and complex structures of the filaments.31 Even recently, whether the existence of additional

10.1021/jp077233y CCC: $40.75 © 2008 American Chemical Society Published on Web 01/01/2008

Disassembly of RecA-dsDNA Filaments ATPγS affects the disassembly of nucleoprotein filament is still discussed.32,33 Therefore, further detailed study of assembly and disassembly of RecA nucleoprotein filament is quite important and interesting. As we know, the native structure of a protein is easily affected by environmental conditions such as solvent, ionic strength, pH, and temperature, and the mechanism of these effects on protein is considerably complex. Protein may become thermodynamically unstable by cooling due to the change of its conformation. This phenomenon is known as cold denaturation, and it is caused by interaction of protein nonpolar groups with water, which is strongly temperature-dependent and is related to the change of enthalpy and entropy that contribute to the Gibbs energy.34,35 Cold denaturation should be a prevalent property for globular proteins, and it is important for studying the unfolded states and folding processes of proteins.36 RecA is a 38-kDa globular protein; thus cold denaturation should have an effect on its native structure. In our study, we attempt to investigate the effect of cold denaturation on the reaction between RecA and dsDNA. We found that the freezing of formed nucleoprotein filament could induce the disassociation of RecA from DNA. To further investigate the influence of solution composition on the stability of the nucleoprotein filaments, after the reaction of RecA and DNA, we changed the composition of the solution before freezing and sample preparation in each assay. Based on this, we compared the different influences of solution composition on the stability of the nucleoprotein filaments through observing the different structures of the products directly by AFM. The experiments indicated the effect of cold denaturation in the interaction of DNA and RecA; on the other hand, through the interaction of DNA and RecA, the effect on the stabilization of RecA could be studied. Experimental Section Reagents. RecA protein (Escherichia coli) was purchased from New England Biolabs at a concentration of 52.85 µM, and was used as received without further purification. λ-DNA (48 502 bp) was purchased from Sino-American Biotechnology Corporation (Beijing, China), and was diluted to 38.7 µM (bp) with 10 mM Tris‚HCl buffer (pH 7.5) before use. ATPγS was purchased from Roche Diagnostics Corporation and dissolved in 10 mM Tris‚HCl buffer (pH 7.5) at a concentration of 7.31 mM. All solutions were prepared using ultrapure water (>18.2 MΩ cm) sterilized at high temperature. Muscovite mica (KAll2(AlSi3)O10(OH)2, V-1 grade) was purchased from Linhe Street Commodity Marketplace (Changchun, China) and was cut into about 1 cm × 1 cm square pieces as AFM substrates. Both sides of the mica surface were freshly cleaved before use. Nucleoprotein Filament Formation Reactions and LowTemperature Treatment. A 15 µL mixed solution (10 mM Tris‚HCl buffer, pH 7.5) containing 2.80 µM RecA, 5.141 µM (bp) λ-DNA, 1.46 mM ATPγS, 6.7 mM Mg(OAc)2, and 100 mM NaCl was incubated at 37 °C for 60 min, which resulted in the formation of RecA-DNA-ATPγS filaments, according to previous work.29 After incubation, 2 µL of the reaction mixture was immediately diluted to 15 µL with 10 mM Tris‚HCl buffer (pH 7.5) containing 7.6 mM Mg2+. In control experiments, 4 °C was chosen as the control temperature. When the same 2 µL of the reaction mixture was diluted to 15 µL, all other conditions were kept the same except that the final diluted solution contained 1 mM 1,4-dithiothreitol (DTT) and 46.7% (v/v)

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Figure 1. Typical AFM images of the RecA-DNA-ATPγS filament (a) and λ-DNA (b) that deposited on the mica directly without other treatment. (z scale is 10 nm.)

glycerol, respectively, in each control experiment. After that, the diluted solution was kept at 4 °C for 4 h for sufficient interaction. Then the solution was further kept at a definite temperature for different times to slow-freeze. AFM Measurements. After the treatment above, the diluted solution was dropped onto a freshly cleaved mica surface, and after remaining there for about 5 min, the mica was gently rinsed with pure water, then dried in air and left sealed for AFM imaging and measurements. All AFM experiments were carried out with a Digital Instruments Nanoscope IIIa (Santa Barbara, CA) in tapping mode. Silicon (Si) cantilevers with spring constants of 0.6-6.0 N/m below their resonance frequency (typically, 67-150 kHz) were used in the measurements. The AFM images were acquired in air under ambient conditions. The value of the set point was regulated to minimize the possible damage to the sample and to maintain the stable image quality at the same time. All AFM images were raw data except for flattening. Results were reported as mean ( standard deviation (number of measurements), and the width of the filament was determined as the full width. Results The structure of nucleoprotein filaments formed by RecA assembling on λ-DNA was imaged by AFM. The architecture of the resulting RecA-DNA-ATPγS nucleoprotein filaments under various conditions was analyzed, and the effects of various solution conditions on the stability of the nucleoprotein filament had been concluded. For convenient discussions, single filaments were taken as examples, except when otherwise indicated. Regular Filaments Formed of RecA on dsDNA. RecA formed regular filaments on linear dsDNA in the presence of ATPγS after a 1 h incubation (Figure 1a). The linear dsDNA was straightened after the RecA protein assembled on it. It was different from the linear dsDNA alone that had many bends along the DNA chain (Figure 1b). This indicated that the formed RecA-DNA-ATPγS filaments had less flexibility than DNA. The nucleoprotein filaments were composed of dots compactly assembled on dsDNA. These dots should be RecA proteins. The average size of the dots was about 20.4 ( 2.4 nm (N ) 105) measured along the axis of the nucleoprotein filaments. Besides that, the average width of the nucleoprotein filaments was about 26.0 ( 1.7 nm (N ) 100) across the filaments. The difference between them might be assigned to the restriction of tip size used in AFM, and both of these data were measured without considering the effect of tip broadening. Because of the compression effect induced by imaging in dry conditions using AFM, the height of single nucleoprotein filaments obtained here was about 2.53 ( 0.3 nm (N ) 100), which was consistent with previous work.29

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Figure 2. Typical AFM images of formed RecA-DNA-ATPγS filaments under various conditions. (a) The formed nucleoprotein filaments were diluted to imaging concentration and kept at 4 °C for 4 h. (b) The formed filaments were further kept at 4 °C for 20 h. (c) After keeping at 4 °C for 4 h, the formed filaments were slow-frozen at -20 °C and were kept for 20 h and then diluted for AFM imaging. (d) RecA was first diluted to the reaction concentration and was stored at -20 °C for about 6 days, and then reacted with DNA. (z scale is 10 nm.)

Effect of Low Temperature on the Nucleoprotein Filaments. Proteins are sensitive to conditions of the surrounding environment such as solvent, ionic strength, pH, and temperature. First, we investigated the structure of RecA-DNAATPγS filaments after they were kept at different temperatures for definite times, respectively (Figure 2). The formed nucleoprotein filaments changed with some small discrepancies, whereas the flexibility of these dissociated filaments nearly had no change and seemed to be consistent with the result above (Figure 1a). In our experiments, 4 °C was taken as the reference comparing with others. After the formed complex was kept at 4 °C for 4 h (referred to as 4 °C-4 h), the nucleoprotein filaments were a little different from the sample prepared immediately (Figure 2a). In Figure 2a, the height of the nucleoprotein filament decreased to about 2.04 ( 0.3 nm (N ) 95), and the average width increased to 50.8 ( 3.9 nm (N ) 95) (including the surrounding lower filmlike substances). Dots assembling along the filaments could be observed. The height of the filmlike substances was about 0.5 nm, and the substances might be composed of dissociated RecA proteins. The further refined structure of the filmlike substances could not be observed because of the resolution of AFM. After keeping at 4 °C for 4 h, the nucleoprotein filaments were further stored at 4 °C for 20 h (referred to as 4 °C-20 h). Most parts of 4 °C-20 h disassembled to 1.21 ( 0.2 nm (N ) 95) in height, and the average width was about 65.9 ( 6.8 nm (N ) 95) (Figure 2b). Many dots that assembled the filaments became lower and smaller than at 4 °C-4 h; meanwhile, the filmlike substances became wider on the AFM image. Moreover, some gaps appeared on the filaments, which suggested that RecA disassembled from DNA and the disassembly slightly happened at different positions along the filament under the experimental conditions. When the filaments were stored at -20 °C for 20 h

Guo et al. (referred to as -20 °C-20 h) instead of 4 °C-20 h, the height of the nucleoprotein filaments obviously decreased to 0.73 ( 0.3 nm (N ) 100) and the average width also increased to 55.5 ( 4.7 nm (N ) 100) (Figure 2c). The filaments seemed to consist of many small dots around the DNA strand, though the dots could not be clearly distinguished due to the resolution of the AFM. These dots might be the dissociated RecA proteins. Some bigger dotlike aggregations on the strands are indicated with an arrow, which might be the aggregations of the dissociated molecules with different sizes and were 4.62 ( 1.3 nm (N ) 35) in height. The dotlike filaments indicated that nucleoprotein filaments greatly disassembled. RecA was first diluted to the reaction concentration and then stored at -20 °C for about 1 week. After that, the diluted RecA reacted with λ-DNA; the resulted filaments also disassembled but were more serious (Figure 2d). Many spherical or elliptical dissociated RecA proteins (the diameter was about 14.4 ( 1.9 nm (N ) 100)) were observed near the remaining RecA-DNA-ATPγS filaments. However, this aggregation state of the RecA protein in AFM images could not be exactly confirmed due to tip forces and tip-shape convolution. Some bigger dots were also found as indicated with the arrow, and they might be nonspecific aggregation of RecA in this situation. Based on the results above, it is presumed that some changes might happen to the RecA in the storing process and result in the disassembly of the nucleoprotein filaments. Effect of Additional DTT on the Nucleoprotein Filaments at Low Temperature. DTT is used to prevent the oxidation of the S-H bond in a protein and to maintain the activity of the protein in preservation and reaction. The effect of DTT on the stabilization of the formed RecA-DNA-ATPγS complexes was also investigated by AFM. When the formed RecA-DNA complexes were diluted with solution containing additional DTT and were kept at 4 °C for 4 h (referred to as DTT-4 °C-4 h), the height of the formed filaments was 2.2 ( 0.4 nm (N ) 105) and the average width was about 53.8 ( 3.1 nm (N ) 105) (Figure 3a). Meanwhile, lower filmlike substances were also observed, which might consist of dissociated proteins around the filaments and did not have very obvious change compared with results at 4 °C-4 h. Hereafter, the following resulted samples were named as additional substance-temperature-time for convenient discussions. After the sample was further kept for 20 h at 4 °C (DTT-4 °C-20 h, Figure 3b), the nucleoprotein filaments were similar to 4 °C-20 h (Figure 2b). The height of the formed filaments was 1.27 ( 0.3 nm (N ) 110), and the average width of the formed filaments was 55.2 ( 3.1 nm (N ) 110); meanwhile dots assembling the filaments became smaller with gaps enlarging between the dots. This indicated that the disassembly was more serious with longer keeping time. Dramatically different filaments were observed when the diluted formed complexes were kept at -20 °C for 20 h (DTT- -20 °C-20 h, Figure 3c). Serious dissociation happened. The nucleoprotein filaments were constituted by small dots with the height of filaments decreasing to 0.87 ( 0.2 nm (N ) 125) and the average width increasing to 58.7 ( 4.5 nm (N ) 125), respectively. This result was similar to -20 °C-20 h (Figure 2c). Therefore, in the cases with additional DTT, the results were similar to the one diluted directly at the same conditions. Effect of Additional Glycerol on the Nucleoprotein Filaments at Low Temperature. Glycerol is used to reduce the polarity of the solution to prevent the protein from being denatured as the protein solution is preserved at low temperature.

Disassembly of RecA-dsDNA Filaments

Figure 3. Typical AFM images of formed RecA-DNA-ATPγS filaments with additional DTT after keeping at different temperatures for different times. (a) The diluted nucleoprotein filaments were kept at 4 °C for 4 h. (b) The diluted nucleoprotein filaments were further kept at 4 °C for 20 h. (c) After keeping at 4 °C for 4 h, the diluted nucleoprotein filaments were slow-frozen at -20 °C and were kept for 20 h. (z scale is 10 nm.)

We also investigated the effect of glycerol on the stabilization of the formed RecA-DNA-ATPγS nucleoprotein filaments by AFM. The formed RecA-DNA-ATPγS filaments became irregular clews and bundles in the presence of glycerol. After the nucleoprotein filaments were kept at 4 °C for 4 h with additional glycerol (glycerol-4 °C-4 h), they were almost integrated filaments in the bundles (Figure 4a), and no obvious disassembly happened. A similar phenomenon was also observed after the nucleoprotein filaments were kept at -20 °C for 20 h (glycerol- 20 °C-20 h, Figure 4c) with additional glycerol. The filaments formed irregular clews. There was a little dissociation of the filaments, and the height of a single filament was 2.00 ( 0.3 nm (N ) 95). These results indicated that the filaments under this condition might be more stable than the examples at -20 °C mentioned above (Figures 2c and 3c). However, obvious disassembly was observed after the complexes were further kept at 4 °C for 20 h (glycerol-4 °C-20 h, Figure 4b), which was similar to DTT- -20 °C-20 h (Figure 3c). The height of the dissociated filaments here was about 0.78 ( 0.3 nm (N ) 105), and many discrete dots were found around the filaments, which might be dissociated proteins. Besides that, Figure 5b further showed at large scale that the formed RecA-DNA-ATPγS nucleoprotein filaments became irregular clews and bundles in the presence of glycerol after keeping for 20 h. However, the nucleoprotein filaments with additional DTT (Figure 5a) were dispersive and did not much intertwist into clews or bundles. Discussion RecA is sensitive to a number of reaction conditions that may affect the conformation of proteins or formed filaments. Different reaction conditions might induce different results such as left-handed superhelices of RecA-dsDNA filaments that were different from the common right-handed superhelices.26

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Figure 4. Typical AFM images of formed RecA-DNA-ATPγS filaments with additional glycerol after keeping at different temperatures for different times. (a) The diluted nucleoprotein filaments were kept at 4 °C for 4 h. (b) The diluted nucleoprotein filaments were further kept at 4 °C for 20 h. (c) After keeping at 4 °C for 4 h, the diluted nucleoprotein filaments were slow-frozen at -20 °C and were kept for 20 h. (z scale is 10 nm.)

Figure 5. Large-scale AFM images of formed RecA-DNA-ATPγS filaments with additional DTT (a) and glycerol (b) after slow-freezing at -20 °C and keeping for 20 h. (z scale is 10 nm.)

It was also discussed whether the absence of additional ATPγS in solution or tip-sample interaction resulted in the disassembly of a single RecA-DNA-ATPγS filament when it was observed using AFM in fluid.32,33 To study the effect of low temperature under different solution conditions in this work, reaction mixtures were deposited onto freshly cleaved mica surfaces and dried in air for AFM measurements. In this situation, the effect of tip-sample interaction can be ignored during the measurement. In this work, when the formed RecA-DNA-ATPγS filaments were kept at 4 °C for 20 h in the presence of additional DTT, the disassemblies were observed (Figure 3b). Compared with DTT-4 °C-4 h (Figure 3a), the height of DTT-4 °C-20 h had some decrease, and it could be observed that the enlarged gaps were distributed on the filaments where RecA had dissociated (Figure 3b). When the formed RecA-DNA-ATPγS filaments were kept at -20 °C for 20 h in the presence of additional DTT (Figure 3c), it could be observed that the height of the filament obviously decreased and the width increased; meanwhile, the disassociated RecA proteins were distributed around the filaments in AFM images. That meant the nucle-

1026 J. Phys. Chem. B, Vol. 112, No. 3, 2008 oprotein filaments disassembled more seriously. It indicated that these disassemblies were resulted from either low temperature or long keeping time, especially low temperature. No matter whether at 4 or -20 °C, the results with additional DTT resembled the ones that were diluted directly (Figure 2). Therefore, the effect of DTT on the stabilization of formed RecA-DNA-ATPγS filaments was not apparent. It was known that DTT could inhibit the oxidation of protein samples, so the results here further indicated that the disassembly was resulted from low temperature and long keeping time, but not from the oxidation of protein. In contrast, when glycerol was used as a substitute for DTT at the same experimental condition of -20 °C, the filaments did not have obvious disassembly (Figure 4c). Glycerol is known to be a common reagent to maintain the conformation of protein at low temperatures such as -20 °C, but the preservation of DNA at low temperatures does not need glycerol. Therefore, the result of filaments with additional glycerol at -20 °C indicated that RecA protein could keep its native structure under this condition. It was further presumed that the disassembly without or with additional DTT was caused by the change of RecA (but not DNA) after keeping at low temperature. This conclusion about RecA protein could be further verified by another control experiment. When RecA was first diluted to the reaction concentration and preserved at -20 °C for a long time, and then reacted with DNA (Figure 2d), the degree of disassembly was on the large side, which was different from -20 °C-20 h (Figure 2c). It was presumed that when the protein was stored at -20 °C for a long time without additional protective reagent, the conformation of RecA might change to a certain extent; therefore, this RecA might not interact with DNA very well and the disassembly might partially happen even in solution. Through these experiments, it was indicated that the change of RecA might weaken the interaction of DNA and RecA in the formed filaments. Besides that, the flexibility of these dissociated filaments seemed to be consistent at low temperature under different experimental conditions. Most of the disassociated RecA proteins were distributed near the filaments, which resulted in the broadened width of the disassembled filaments. The width was nearly uniform, but almost no bare DNA strands were found. This showed that the formed RecA-DNA-ATPγS filaments did not have a biggish change in solution and the disassembly mainly occurred in the process of deposition on the mica surface. The disassembly might result from the interaction of nucleoprotein filaments and mica; otherwise bare DNA strands would be observed as results. We deduce the possible mechanism as follows. We have proven that the conditions of low temperature and others might weaken the interaction of protein and DNA, which might result in the change of the formed nucleoprotein filament caused by some ambient condition. According to the previous report,37 the counterion concentration near the highly negatively charged mica surface was high with the lower concentration of monovalent salt. When the formed protein/DNA complexes approached the mica surface, the assembly-disassembly equilibrium shifted toward the one that dominated by high ionic strengths, and brought on corresponding changes. In our experiments, the concentration of NaCl in the imaging solution was about 13.3 mM, which was lower than the counterion concentration near the mica surface. Since the protein-DNA interaction of the formed filaments has already weakened, the filaments should disassemble easily when they approach the mica surface in the preparation process of AFM samples because of high counterion

Guo et al. concentration. When the complexes were fully adsorbed on the surface, the situation was different. As previously reported, the presence of the concentrated counterion layer on the mica surface could induce strong surface friction,38 which could slow the diffusion of the filaments after they were fully adsorbed on the mica surface. Therefore, the dissociated RecA proteins were observed to be distributed around the filament with definite width. Despite all this, the process was complicated, and it needed further study. Another phenomenon referred to in our experiments was the abnormal effect of glycerol on nucleoprotein filaments at 4 °C for 20 h. After further keeping at 4 °C for 20 h, the sample with additional DTT disassembled partially (Figure 3b). However, the sample with glycerol disassembled (Figure 4b) obviously, which was similar to the example with additional DTT at -20 °C for 20 h (Figure 3c). The reason was still confused, and the possible explanation was that, though glycerol could reduce the polarity of the solution to prevent the protein from being denatured at low temperatures such as -20 °C, it might not stabilize the natural RecA morphology when the nucleoprotein filaments were kept at 4 °C for 20 h. Therefore, the formed nucleoprotein filaments disassembled after keeping at 4 °C for 20 h. The reason for this phenomenon needs further study. Besides the results mentioned above, when the samples were measured at large scale, it was observed that the formed RecA-DNA-ATPγS nucleoprotein filaments became irregular clews and bundles in the presence of glycerol (Figure 5b). However, the nucleoprotein filaments with additional DTT (Figure 5a) were obviously different. The conceivable interpretation was also that the added glycerol could weaken the polarity of the solution, which decreased the repulsive force between RecA molecules or facilitated the interaction of RecA. Conclusion In summary, we observed the disassembly of formed filaments at different experimental conditions by AFM. Cold denaturation could induce the instability and disassembly of the nucleoprotein filaments. Furthermore, when the formed filaments were kept at -20 °C for 20 h with additional DTT, the nucleoprotein filaments disassembled and consisted of small spherical molecules. The height of the filaments decreased and the width increased, resembling the example without anything added. In contrast, the formed filaments did not disassemble at the same experimental condition except for the addition replaced by glycerol. On the other hand, the results at 4 °C after keeping for 20 h were different. The disassembly with additional DTT was not obvious, but disassembly was observed with additional glycerol. The differences at the different temperatures were mainly due to the change of RecA after keeping at low temperature for a comparatively long time. This change might weaken the interaction of DNA and RecA in the formed filaments. Thus, in the process of deposition onto the mica surface, the RecA protein disassembled from the nucleoprotein filaments because of the interaction between filaments and the mica surface. Furthermore, irregular clews and bundles were observed in the presence of glycerol. This was mainly attributed to the weakened polarity of the solution through adding glycerol that decreased the repulsive force between RecA molecules or facilitated the interaction of RecA. This information may be helpful for understanding the relevant function of influencing factors on RecA and DNA in further theoretical studies, and may be valuable for studying the RecA-DNA interaction by using AFM.

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