NANO LETTERS
Atomic Force Microscopy Can Detect the Binding of Yeast Replication Factor C to DNA
2003 Vol. 3, No. 1 39-41
Lisa Green,† Mark Schotanus,† Michael A. McAlear,‡ and Elizabeth A. Howell* Department of Biology, CalVin College, Grand Rapids, Michigan 49546, and Department of Molecular Biology and Biochemistry, Wesleyan UniVersity, Middletown, Connecticut 06495 Received October 4, 2002; Revised Manuscript Received November 15, 2002
ABSTRACT Both DNA replication and DNA repair processes depend on the activity of numerous DNA polymerase accessory proteins. For example, the eukaryotic clamp loader replication factor C (RFC) is required to load the sliding DNA clamp, PCNA, onto DNA at primer/template junctions. Although the complete RFC-catalyzed loading reaction requires ATP hydrolysis, indirect assays such as surface plasmon resonance have indicated that RFC can bind to single-stranded DNA in the absence of ATP. We have used atomic force microscopy to verify directly that RFC from Saccharomyces cerevisiae can bind to single-stranded DNA in the absence of nucleotides.
Introduction. The growth and survival of all organisms is dependent on the effective and efficient duplication and maintenance of the organism’s genome. The complex processes of DNA replication and DNA repair require the activity of several DNA polymerases and their associated proteins. In eukaryotes, the DNA replication machinery includes DNA polymerases R, δ, and as well as topoisomerases, single-stranded DNA binding proteins, and the cofactors replication factor C (RFC) and proliferating cell nuclear antigen (PCNA).1 In particular, RFC and PCNA work together to enhance the processivity of DNA polymerases. PCNA acts as a sliding clamp that can tether DNA polymerases to the DNA template, allowing DNA polymerase to replicate DNA with greater efficiency.2 RFC is a heteropentameric protein that operates as a clamp loader, enabling PCNA to encircle DNA.3-5 In addition to loading PCNA onto DNA, RFC is also fundamental to the switch from polymerase R to polymerase δ that is necessary for processive DNA replication. After pol R, or primase, synthesizes the primer, it is displaced from the template as RFC binds to DNA. RFC then loads PCNA and pol δ onto the DNA at the primer-template junction.6 The primer-template junction of the replication fork consists of both double-stranded and single-stranded regions. There is evidence that RFC selectively binds to primed DNA;7 however, recent studies have shown a preference of RFC to bind single-stranded DNA.8 We investigated the * Corresponding author. E-mail:
[email protected]. Phone: (616) 9577085. † Calvin College. ‡ Wesleyan University. 10.1021/nl025831c CCC: $25.00 Published on Web 12/14/2002
© 2003 American Chemical Society
ability of RFC to bind to single-stranded DNA by using the atomic force microscopy. Atomic force microscope (AFM) is a current and useful technique for studying DNA and proteins in their natural state.9-13 AFM uses either air or liquid conditions to image DNA and other molecules on the nanometer scale. Unlike other forms of microscopy, AFM allows for the imaging of samples without staining or fixing. Images are produced via contours in the sample’s surface that are translated into a topographical map and in turn into an image. Thus, AFM is advantageous because of its ability to detect not only the intricacies of DNA but also proteins such as Pho4 and Mig1,14 photolyase,12 and RecA15 interacting with DNA in their native conditions. Therefore, we reasoned that AFM would be a valuable technique to further examine the Saccharomyces cereVisiae clamp loader (scRFC) and its interactions with single-stranded DNA. The formation of the RFC-PCNA-DNA complex requires ATP, and the dissociation of RFC upon PCNA binding occurs because of the hydrolysis of ATP.16 The five different subunits of the RFC protein have been analyzed for their ATPase activity; Cai et al. have found that the three subunit complex of p40‚p37‚p36 is a DNA-dependent ATPase. In addition, of the five subunits, only RFC5 does not contain an ATP binding domain.2 AFM experiments done with ATP and RFC have shown RFC undergoing a conformational change in the presence of ATP. RFC remains in a more closed or U shape in the absence of ATP but conforms to an open structure in the presence of ATP. The addition of the different nucleotide analogues ADP and the nonhydrolyzable ATPγS, however, showed minimal transforma-
tions in the shape of RFC.17 Keller et al. have shown RFC binding to single-stranded DNA in the presence of ATP using electron microscopy.8 In addition, the surface plasmon resonance technique has revealed the binding of RFC to single-stranded DNA in the absence of ATP and in the presence of ATP, ATPγS, and ADP.2 In this paper, we use atomic force microscopy to verify directly the ability of scRFC to bind to single-stranded DNA in the absence of nucleotide as well as in the presence of ATP, ATPγS, and ADP. Materials and Methods. DNA. The DNA used in all of the experiments was M13mp18 single-stranded DNA (New England Bio Labs, Beverly, MA). Preparation of Functionalized APS-Mica. Mica disks were prepared daily for each experiment by affixing ruby mica disks (SPI supplies, West Chester, PA) onto 15-mm steel disks (Ted Pella, Inc., Redding, CA) with double-sided tape. The mica disks were freshly cleaved with one-sided tape to remove any residue, thus providing a flat surface. The freshly cleaved disks were functionalized by pipetting 100 µL of 0.001% (v/v in ultrapure water with a resistance greater than 18 MΩ cm) 3-aminopropyltriethoxysilane (APS) solution onto the center of the mica disc.18,19 The disks were then incubated under glass for 20 min at room temperature. After the incubation time, AP-mica surfaces were rinsed five times with 1-mL portions of ultrapure water and then dried under a gentle stream of air. The 0.001% (v/v in ultrapure water) APS solution was made by using a syringe, because of its air sensitivity, to draw out 1 µL of 3-aminopropyltriethoxysilane (Aldrich Chemical Co., Inc., Milwaukee, WI), which was then added to 100 mL of ultrapure water. Both the 3-aminopropyltriethoxysilane and the 0.001% APS were stored in a desiccator. Purification of Recombinant Yeast RFC. Recombinant yeast RFC was overproduced in an E. coli strain harboring a plasmid bearing all five of the yeast RFC genes under inducible GAL promoters. Ten liters of the E. coli strain was grown up, and the RFC was purified by Affigel Blue, PCNA-agarose, and MonoS chromatography as described previously.20 RFC Binding Assay. All of the solutions used for AFM sample preparation were filtered upon preparation with a 0.22-µm filter. The standard RFC binding assay consisted of 200 nM RFC, 4 mM Tris‚HCl (pH 7.5), 1.6 mM MgCl2, 0.2 mM DTT, 20 mM NaCl, and 1.5 µg DNA brought to a final volume of 12 µL with ultrapure water. After mixing the components, the samples were kept on ice for 10 min. After the incubation on ice, 1:2 dilutions of each sample were made using freshly filtered incubation buffer (20 mM Tris‚ HCL pH 7.5, 8 mM MgCl2, 4% glycerol, 0.5 mM EDTA, 2 mM DTT, and 50 mM NaCl in ultrapure water). The 1:2 diluted samples were then incubated for 10 min at 37 °C. After incubation, the samples were kept for an additional 1 min on ice before they were loaded onto the mica disks. In assays requiring nucleotide, 1 mM ATP, 1 mM ATPγS (NEN, made fresh daily), or 1 mM ADP was added at the start of the binding assay. In the assay with bovine serum 40
albumin (BSA), boiled BSA was added in place of scRFC to a final concentration of 13 µg/mL. DNA/Sample Immobilization on APS-Mica. The samples were immobilized on APS-mica by pipetting 1 µL of the desired sample onto the center of the APS-mica disk. The sample-laden disk was then incubated under glass for 10 min at room temperature. Unbound DNA and protein were then washed off by pipetting 1 mL of ultrapure water onto the surface and then repipetting the water off of the mica surface. This was repeated using a second 1 mL of ultrapure water. After drying the surface with a gentle stream of air, the sample was prepared for imaging. Imaging of Samples Using Atomic Force Microscopy (AFM). To image the samples, a multimode SPM NanoScope IIIa (Digital Instruments, Santa Barbara, CA) in tapping mode was used. The NanoScope III version 4.43r8 software was used to capture the images. The AFM images were initially scanned at a 5-µm scan size with a scan rate of 1 Hz and height of 5 nm and then zoomed in as necessary. The tips that were used were TESP NANOPROBE SPM tips for di scanning probe microscopes. Once the tip was lowered on the surface of the sample, it was auto tuned, engaged, disengaged, and then re-auto tuned to ensure the appropriate drive amplitude. Increasing the amplitude set point until the image was lost and then decreasing the set point value four times allowed us to find the appropriate amplitude set point. Data Analysis. The images captured were then analyzed using NanoScope III version 4.43r8 software. For each different assay repeated on multiple days, 5-µm scan-size images from the different days were used. The center of the image was zoomed in to a 1.98-µm size. Using the section function in the NanoScope software, all of the particles bound to DNA in the 1.98-µm area were measured for their height in reference to the surface of the mica. For every assay, roughly 100 particles were analyzed; the heights were averaged and given a calculated standard deviation. Results. To visualize DNA bound with scRFC, we performed a modified RFC binding assay. Heteropentameric RFC from the budding yeast S. cereVisiae was incubated with single-stranded DNA (M13 mp 18) in the presence of binding buffer for 10 min on ice, and then the reaction was diluted 1:2 into incubation buffer with or without nucleotide. Following a 10-min incubation at 37 °C, samples were loaded onto mica disks that had been functionalized with a 0.001% 3-aminopropyltriethoxysilane (APS) solution. The use of APS-mica greatly facilitates the binding of negatively charged DNA to negatively charged mica.18,19 When samples containing DNA alone were imaged, naked single-stranded DNA molecules were clearly visible. (Figure 1a). In contrast, when samples containing DNA and scRFC were imaged, the large majority of DNA molecules were bound with scRFC, with a minimal background of unbound protein (Figure 1b). The bound protein is most likely to be scRFC. Its uniform appearance and the clarity of the surrounding mica surface suggest that the bound molecule is a protein rather than background contamination such as dust. The only other protein in the reaction, bovine serum albumin present in the scRFC reconstitution buffer, does not bind specifically to Nano Lett., Vol. 3, No. 1, 2003
Figure 1. Replication factor C binds single-stranded DNA. Singlestranded DNA molecules were imaged by tapping-mode AFM (a) in the absence of protein, (b) in the presence of scRFC, or (c) in the presence of bovine serum albumin. Image size is 1 µm × 1 µm × 5 nm.
in the presence of ATP, ATPγS, and ADP. This technique will next be applied to the visualization of yeast RFC in the presence of nicked DNA to examine directly the ability of the protein to bind to a region of DNA that mimics the primer-template junction. In this manner, the data obtained by atomic force microscopy will further complement that found by more indirect methods such as surface plasmon resonance and will extend our knowledge of the function of the eukaryotic DNA replication complex. Acknowledgment. We thank Kumar Sinniah for extensive advice in experimental design and manuscript preparation. This work was supported by Grant 52002664 from the Howard Hughes Medical Institute to Calvin College and by Grant GM54818 (to M.M.) from the National Institutes of Health. We also acknowledge support from the National Science Foundation (Grant CHE 9871225) to purchase a scanning probe microscope. References
Figure 2. Histogram of the frequency of a particle bound to DNA vs the size of the particle in nanometers.
DNA (Figure 1c). In addition, the size of the particles is consistent with that of scRFC. ScRFC has a molecular mass of 249 kD; the highly homologous human RFC has a molecular mass of 290 kD, and its approximate size is calculated to be 16 × 14 nm.17 Given that the tip will likely compress the protein and that imaging proteins in air may result in as much as a 5-7-fold compression,15 the average height of the bound protein (5.2 ( 2 nm, n ) 106, Figure 2) appears to be reasonable. As a point of reference, the average height of DNA molecules in our sample was found to be 0.8 nm, which is agreement with the 1-nm height found for single-stranded DNA imaged in air.10 Although human RFC has been visualized by electron microscopy to bind single-stranded DNA in an ATPdependent manner,8 our data verifies scRFC binding in the absence of any nucleotides (Figure 1b). ScRFC was also bound to single-stranded DNA in the presence of 1 mM ATP, 1 mM ATPγS, and 1 mM ADP (data not shown). This data correlates well with the surface plasmon resonance assays that demonstrated the ability of RFC to bind single-stranded DNA in the absence of nucleotides and in the presence of ATP, ATPγS, and ADP, as shown by Gomes and Burgers.2 In conclusion, we have used the atomic force microscope to visualize the heteropentameric replication factor C protein from budding yeast binding to single-stranded DNA. The protein was bound in the absence of nucleotides as well as
Nano Lett., Vol. 3, No. 1, 2003
(1) Burgers, P. M. J. In Eukaryotic DNA Replication; Blow, J. J., Ed.; IRL Press: New York, 1996; pp 1-28. (2) Gomes, X. V.; Burgers, P. M. J. J. Biol. Chem. 2001, 276, 34769. (3) Tsurimoto, T.; Stillman, B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 1023. (4) Podust, V. N.; Tiwari, N.; Stephan, S.; Fanning, E. J. Biol. Chem. 1998, 273, 31992. (5) Zhang, G.; Gibbs, E.; Kelman, Z.; O’Donnell, M.; Hurwitz, J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 1869. (6) Maga, G.; Stucki, M.; Spadari, S.; Hu¨bscher, U. J. Mol. Biol. 2000, 295, 791. (7) Cai, J.; Uhlmann, F.; Gibbs, E.; Flores-Rozas, H.; Lee, C.-G.; Phillips, B.; Finkelstein, J.; Yao, N.; O’Donnell, M.; Hurwitz, J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12896. (8) Keller, R. C.; Mossi, R.; Maga, G.; Wellinger, R. E.; Hu¨bscher, U.; Sogo, J. M. Nucleic Acids Res. 1999, 27, 3433. (9) Hansma, H. G.; Laney, D. E.; Bezanilla, M.; Sinsheimer, R. L.; Hansma, P. K. Biophysical Journal. 1995, 68, 1672. (10) Hansma, H. G.; Revenko, I.; Kim, K.; Laney, D. E. Nucleic Acids Res. 1996, 24, 713. (11) Mu¨ller, D. J.; Anderson, K. Trends Biotechnol. 2002, 20, S45. (12) van Noort, S. J. T.; van der Werf, K. O.; Eker, A. P. M.; Wyman, C.; de Grooth, B. G.; van Hulst, N. F.; Greve, J. Biophys. J. 1998, 74, 2840. (13) Yokota, H.; Nickerson, D. A.; Trask, B. J.; van den Engh, G.; Hirst, M.; Sadowski, I.; Aebersold, R. Anal. Biochem. 1998, 264, 158. (14) Moreno-Herrero, F.; Herrero, P.; Colchero, J.; Baro´, A. M.; Moreno, F. Biochem. Biophys. Res. Commun. 2001, 280, 151. (15) Umemura, K.; Ikawa, S.; Nishinaka, T.; Shibata, T.; Kuroda, R. Nucleic Acids Symp. Ser. 1999, 42, 235. (16) Cai, J.; Yao, N.; Gibbs, E.; Finkelstein, J.; Phillips, B.; O’Donnell, M.; Hurwitz, J. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11607. (17) Shiomi, Y.; Usukura, J.; Masamura, Y.; Takeyasu, K.; Nakayama, Y.; Obuse, C.; Yoshikawa, H.; Tsurimoto, T. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14127. (18) Lyubchenko, Y. L.; Gall, A. A.; Shlyakhtenko, L. S. Methods Mol. Biol. 2001, 148, 569. (19) Umemura, K.; Ishikawa, M.; Kuroda, R. Anal. Biochem. 2001, 290, 232. (20) Gomes, X. V.; Gary, S. L.; Burgers, P. M. J. Biol. Chem. 2000, 275, 14541.
NL025831C
41
