Anal. Chem. 2000, 72, 3138-3141
MutS-Mediated Detection of DNA Mismatches Using Atomic Force Microscopy Hui Bin Sun and Hiroki Yokota*
Biomedical Engineering Program, Departments of Mechanical Engineering and of Anatomy and Cell Biology, Indiana University Purdue University at Indianapolis, Indianapolis, Indiana 46202
We have developed an atomic force microscopy-based method for detecting DNA base-pair mismatches using MutS protein isolated from E. coli. MutS is a biological sensor and a locator of DNA base-pair mismatches. It binds specifically to a mismatched DNA base pair and initiates a process of DNA repair. To test the possibility of visually detecting mismatched base pairs by atomic force microscopy, we prepared DNA templates ∼500 bp in length consisting of a single or multiple base-pair mismatches. We demonstrate that MutS binding sites on individual DNA molecules were readily detectable by atomic force microscopy and that the observed positions were in good agreement with the predicted sites of basepair mismatches at a few-nanometer resolution. The technique described here is rapid and sensitive and is expected to be useful in screening mutations and DNA polymorphisms. The completion of the human genome project in 2003 will provide a unique opportunity for studying genetic mutations and DNA polymorphisms in humans.1,2 DNA sequence alterations are the primary cause for genetic disorders, and an understanding of the relationship between sequence variations and disease risk is critical to disease prevention and clinical treatments in the post human genome sequence era. Furthermore, detection of sequence alterations provides an unambiguous method for identifying individuals in forensic situations and in questions of parenthood.3,4 To facilitate access to human DNA information, technologies are needed that rapidly compare similarities and differences between various DNA samples. Since many genetic disorders are caused by a point mutations and the most common polymorphisms in the human genome are single base-pair differences, a reliable screening tool for detecting mismatched base pairs would be valuable.5 * Corresponding author: Department of Anatomy and Cell Biology, Indiana University School of Medicine, 635 Barnhill Dr., MS-504, Indianapolis, IN 46202. Phone: 317-274-2448. Fax: 317-278-2040. E-mail:
[email protected]. (1) Collins, F. S.; Patrinos, A.; Jordan, E.; Chakravarti, A.; Gesteland, R.; Walters, L. Members of the DOE and NIH planning groups. Science 1998, 282, 682-689. (2) Marshall, E. Science 1999, 284, 1439-1440. (3) Dino-Simonin, N.; Brandt-Casadevall, C. Forensic Sci. Int. 1996, 81, 6172. (4) Schneider, P. M. Forensic Sci. Int. 1997, 88, 17-22. (5) Kruglyak, L. Nat. Genet. 1999, 22, 139-144.
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Biological organisms possess built-in machinery for DNA mismatch repair.6,7 In Escherichia coli, for instance, one of the DNA repair pathways is initiated by the binding of a 97-kDa MutS protein to a site of DNA damage, i.e., mismatched DNA base pairs. After the binding of MutS, enzymes such as DNA helicase and DNA polymerase work synergistically and repair the damaged DNA. Although the binding affinity of MutS to eight different mismatches, such as (A/A), (A/C), and (A/G), apparently varies, the formation of MutS-DNA complexes has been reported to be specific to all base-pair mismatches by electrophoretic mobility shift assays or molecular images taken by electron microscopy.8-10 In this report, we describe a MutS-mediated method for detection of DNA base-pair mismatches by using atomic force microscopy (AFM).11 AFM is widely used to capture structural images at nanometer resolution of biological molecules including nucleic acids, proteins, and membranes.12-15 In analyzing DNA sequence variations, AFM enables us to characterize individual DNA molecules longer than the molecules commonly used for electrophoretic mobility shift assay, DNase footprinting, or microfabricated DNA arrays. Previously we developed two AFMbased methods for straightening and immobilizing DNA and protein-DNA complexes on an atomically flat mica surface.16-18 We applied a similar method for sample preparation and demonstrate here for the first time that MutS-DNA complexes are detectable by AFM and the observed binding sites of MutS are (6) Modrich P. Annu. Rev. Genet. 1991, 25, 229-253. (7) Kolodner, R. D.; Marsischky, G. T. Curr. Opin. Genet. Dev. 1999, 9, 8996. (8) Parsons, B. L.; Heflich, R. H. Mutat. Res. 1997, 374, 277-285. (9) Bellanne-Chantelot, C.; Beaufils, S.; hourdel, V.; Lesage, S.; Morel, V.; Dessinais, N.; Le Gall, I.; Cohen, D.; Dausset, J. Mutat. Res. Genomics 1997, 382, 35-43. (10) Gotoh, M.; Hasebe, M.; Ohira, T.; Hasegawa, Y.; Shinohara, Y.; Sota, H.; Nakao, J.; Tosu, M. Genet. Anal.: Biomol. Eng. 1997, 14, 47-50. (11) Binnig, G.; Quate, C. F.; Gerber C. Phys. Rev. Lett. 1986, 56, 930-933. (12) Hansma, P. K.; Elings, V. B.; Marti, O.; Bracker, C. E. Science 1988, 242, 209-242. (13) Bustamante, C.; Vesenka, J.; Tang, C. L.; Rees, W.; Guthold, M.; Keller, R. Biochemistry 1992, 31, 22-26. (14) Radmacher, M,; Fritz, M.; Hansma, H. G.; Hansma, P. K. Science 1994, 265, 1577-1579. (15) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 265, 415-417. (16) Yokota, H.; Nickerson, D. A.; Trask, B. J.; van den Engh, G.; Hirst, M.; Sadowski, I.; Aebersold, R. Anal. Biochem. 1998, 264, 158-164. (17) Yokota, H.; Fung, K.; Trask, B. J.; van den Engh, G.; Sarikaya, M.; Aebersold, R. Anal. Chem. 1999, 71, 1663-1667. (18) Yokota, H.; Sunwoo, J.; Sarikaya, M.; van den Engh, G.; Aebersold, R. Anal. Chem. 1999, 71, 4418-4422. 10.1021/ac991263i CCC: $19.00
© 2000 American Chemical Society Published on Web 06/16/2000
Figure 1. The 449-bp DNA templates used in this study. Template 1 consisted of a single base-pair mismatch of (T/C) or (G/A) at a site 137 bp apart from one end. Template 2 consisted of a single basepair mismatch of (C/A) or (T/G) at a site 182 bp apart from the same end. Template 3 consisted of two base-pair mismatches in the templates 1 and 2.
Figure 2. AFM height image of MutS-DNA complexes. Height is indicated by a color code with dark (0 nm) and light (5 nm). (A-C) The MutS-DNA complexes corresponding to template 1. The specific binding site of MutS was predicted at 0.305 (137 bp away from one end of the 449-bp DNA template). (D-E) The MutS-DNA complexes corresponding to template 2. The specific binding site of MutS was predicted at 0.405 (182 bp away from one end of the 449-bp DNA template).
in good agreement with the predicted location of mismatched DNA base pairs. MATERIALS AND METHODS DNA Preparation. Six DNA templates were prepared by synthesizing custom-made oligonucleotides, cloning them in a plasmid, and hybridizing a pair of nucleotides consisting of one or two base-pair mismatches (Figure 1). Briefly, three custommade oligonucleotides 100 nucleotides long were first amplified by PCR, and the PCR products were digested with SacI and XhoI to obtain 83-bp DNA fragments. The DNA fragments were then inserted into the multiple cloning site of the plasmid (pBluescript II KS, Stratagene), and the plasmids amplified in E. coli were isolated using a plasmid isolation kit (Qiagen). After digestion of the isolated plasmids by PvuII, the 449-bp DNA fragments used in this study were recovered from a 1% agarose gel and purified using a gel extraction kit (Qiagen). A pair of different DNA clones were denatured and hybridized to form DNA templates containing one or two base-pair mismatches. Assuming a random pairing of 449-nt DNA strands, half of the DNA duplexes were expected to be heteroduplexes. Templates 1 and 2 were designed to consist of a one base-pair mismatch, and template 3 was constructed to consist of two base-pair mismatches. Formation of MutS-Heteroduplex DNA Complexes. The DNA duplexes, half of them consisting of one or two base-pair mismatches, were each suspended in a 10 µL of buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM KCl, and 2 mM MgCl2. To allow the formation of MutS-heteroduplex DNA complexes, 1 µL of a thermostable MutS suspension (Epicenter Technologies Corp.) at 100 ng/µL was added to the DNA suspension and the mixture was incubated at 22 °C for 30 min. The mixture of DNA and MutS was then spread on a freshly cleaved mica sheet (25 mm × 25 mm, Ted Pella Inc.) by using a
Figure 3. Histogram showing the normalized location of the bound MutS on DNA templates. (A) Histogram corresponding to template 1. The mean and the standard deviation of the normalized binding position were determined as 0.303 ( 0.018 (N ) 36), while the predicted binding site was 0.305. (B) Histogram corresponding to template 3. The mean and the standard deviation for two binding sites were 0.295 ( 0.022 (N ) 15) and 0.398 ( 0.018 (N ) 32), while the predicted sites were 0.305 and 0.405.
DNA spin-stretcher as described previously.17 Briefly, a mica sheet was mounted horizontally on the spin-stretcher using a doublesided tape, and the mica plate was spun at ∼5000 rpm. A series of droplets was gently dispensed on the spinning center with a pipet, in the following order and with ∼30-s intervals: 50 µL of H2O for prerinsing, 50 µL of 500 mM MgCl2 solution for precoating the surface, 50 µL of H2O for rinsing, 10 µL of the sample solution, and 50 µL of H2O for postrinsing the sample surface. Imaging by Atomic Force Microscopy. A Nanoscope III atomic force microscope (Digital Instruments, Inc.) was used to capture images of MutS-heteroduplex DNA complexes immobilized on the mica surface. AFM was performed in the ambient air at 15-20% humidity. The tapping mode was used to reduce Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
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Figure 4. AFM height image of single-MutS-DNA complex and double-MutS-DNA complex. To present 3-D structural features, the images are displayed with a surface tilt of 20°. The white arrow indicates the bound MutS. (A) Single-MutS-DNA complex corresponding to template 1. (B) Double-MutS-DNA complex corresponding to template 3.
any damage to biological samples caused by physical contact with the tip. The tapping frequency chosen was ∼200 kHz, a frequency near the resonance of the cantilever. A scan field of view was set to 2 µm × 2 µm with the scanning rate of 0.5-1 Hz and 512 scanning lines. The silicon tips we used had an estimated curvature of 10-20 nm. The height bar was linear between 0 and 5 nm. Height images were flattened to remove the background curvature of the mica surface, and the images were analyzed using NIH Image 1.60 image analysis software. The normalized MutS binding position was defined as d1/(d1 + d2), where d1 and d2 (d1 < d2) are the length of DNA segments bisected by MutS. RESULTS Detection of MutS-DNA Complex. To determine proper biochemical conditions for detection of MutS-DNA complexes by AFM, we investigated the effect of the DNA concentration and the MutS concentration on the retention of MutS-DNA complexes on the mica surface. Although a variation existed from one mica surface to another, an increase in the MutS concentration enhanced the formation of MutS-DNA complexes but it reduced the retention of target molecules on the surface. In this report, 10 ng of DNA templates was incubated with 20 ng of 97-kDa MutS in 10 µL of buffer. The ratio of MutS protein to DNA templates was estimated to be ∼6. In a typical 2 µm × 2 µm field of view, 20-30 DNA molecules were detectable, and roughly 10% of the DNA in the field was bound by MutS (Figure 2). Note that half of the duplex DNA we used was expected to form a homoduplex with no base-pair mismatch. We did not observe more than one MutS bound to template 1 or 2, which consisted of a single basepair mismatch. Determination of Specific MutS Binding Sites. To examine the specificity of the binding of MutS to DNA base-pair mismatches, we analyzed the distribution of 83 binding sites on 3140
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templates 1 and 3. A normalized site value between 0 and 0.5 was assigned to each binding site by determining d1/(d1 + d2), where d1 and d2 (d1 < d2) corresponded to two DNA segments bisected by MutS. Figure 3 displays two such histograms for the templates 1 and 3, respectively. Each histogram represents the observation made for a single incubation preparation. The mean and the standard deviation of the binding sites on the template 1 were determined as 0.303 ( 0.018 (N ) 36, sample number) with good agreement with the predicted value of 0.305. For template 3 consisting of two base-pair mismatches, two local maximums appeared in the observed distribution at 0.295 ( 0.022 (mean ( standard deviation, N ) 15) and 0.398 ( 0.018 (mean ( standard deviation, N ) 32). The observed site values were in good agreement with the predicted value of 0.305 and 0.405. The normalized positional difference between the observation and the prediction ranged from 0.002 to 0.01. Since all DNA templates were 449 bp long, the maximum positional difference corresponded to 4-5 bp or 1-2 nm. In the same binding assay using the template 3, MutS was bound 2.1 times as frequently to the mismatch site of (C/A) or (T/G) as to the other site of (T/C) or (G/A). Detection of Double-MutS-DNA Complexes. We next examined whether MutS proteins, tandemly bound to two point mismatches separated by 45 bp in template 3, would be identifiable as two proteins by AFM. We observed that ∼10% of the DNA molecules on the mica surface formed MutS-DNA complexes, and that ∼1% of all DNA templates exhibited a structure bound to DNA with two globular parts (Figure 4). Compared to MutS protein singly bound to template 1, an apparent pair of MutS proteins on template 3 exhibited an elongated conformation along DNA. In determining the size of the bound MutS, we defined their outer edge using the height identical to the height of DNA. The mean and the standard deviation of the bound MutS were
determined as 19.0 ( 0.3 × 15.0 ( 0.2 (major axis × minor axis, N ) 20). In Figure 4, the single MutS footprint on DNA was measured as 19 nm × 15 nm (major axis × minor axis), while the footprint of apparent double MutS was 26 nm × 15 nm (major axis × minor axis) with a constriction in the middle. DISCUSSION We describe a rapid and sensitive MutS-mediated method of identifying the position of DNA base-pair mismatches using AFM. For 449-bp DNA templates consisting of one or two base-pair mismatches, the site of mismatches was determined from individual MutS binding sites within a few-nanometer deviation. The observed binding of MutS to base-pair mismatches was specific in our AFM-based assay. The difference between the observed site and the prediction was less than 5 bp (2 nm) for all three cases studied in this report, and the standard deviation was 8-9 bp (∼3 nm) in the distribution of 15-36 MutS binding sites. A pair of MutS proteins, apparently bound tandemly at two binding sites separated by 45 bp, was identifiable from a shape resembling a cluster of two globular structures. The measurement was close enough to locate DNA mismatches within a particular genomic region, and stretching DNA molecules enabled to yield good precision. AFM is becoming a useful tool for analyzing genomic DNAs mapping a recognition site of a restriction enzyme or constructing a physical DNA map of protein-binding sites.19-21 Unlike conventional molecular tools such as electrophoretic mobility shift assays or DNase footprinting, the AFM-based method allows us to use large DNA templates from a minute amount of sample without any modification or labeling of protein or DNA molecules. DNA molecules over 100 kbp in length can easily be straightened by the stretching apparatus we have developed and determining sites (19) Allison, D. P.; Kerper, P. S.; Doktycz, M. J.; Spain, J. A.; Modrich, P.; Larimer, F. W.; Thundat, T.; Warmack, R. J. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 8826-8829. (20) Allison, D. P.; Kerper, P. S.; Doktycz, M. J.; Thundat, T.; Modrich, P.; Larimer, F. W.; Johnson, D. K.; Hoyt, P. R.; Mucenski, M. L.; Warmack, R. J. Genomics 1997, 41, 379-384. (21) Wyman, C.; Rombel, I.; North, A. K.; Bustamante, C.; Kustu, S. Science 1997, 275, 1658-1661. (22) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; and Fodor, S. A. Science 1996, 274, 610614. (23) Wang D. G.; et al. Science 1998, 280, 1077-1082.
of bound MutS along the uncoiled DNA is straightforward. Since neither end of the DNA molecules was labeled in this report, the site of MutS binding had a directional ambiguity. It is possible to remove this ambiguity by including a reference base-pair mismatch close to one end. Understanding variations of affinity and specificity among eight possible mismatches will help interpret a distribution of MutSbinding sites. In template 3, for instance, four different kinds of mismatches, such as (T/C), (G/A), (C/A), and (T/G), were included at an equal frequency. One pair of (T/C) or (G/A) mismatch, which was positioned at 137 bp from one end, gave a fewer number of MutS-DNA complexes than the other pair of (C/A) or (T/G) mismatch at 182 bp away from the same end. In a study with the surface plasmon resonance sensor, base mismatch such as (T/C) or (T/T) was reported to exhibit lower affinity to MutS than a mismatch such as (G/A) or (G/T).10 An AFM-based affinity study using DNA templates consisting of one kind of mismatch only will help further characterize the described MutS-mediated approach. In conclusion, we have described a MutS-mediated method of detecting DNA base-pair mismatches by AFM. In the post human genome sequence era, identifying similarities and differences among DNA samples is expected to be a growing task. The described method provides a rapid and sensitive tool for identifying differences by placing a landmark at a site of base-pair mismatches at nanometer resolution. This method is expected to complement currently available hybridization-based methods represented by DNA chip technologies, most of which are designed to detect base-pair matches of relatively short oligonucleotides.22,23 ACKNOWLEDGMENT We appreciate Satomi Ohnishi (National Institute of Materials and Chemical Research, Japan), Helen Hansma (University of California, Santa Barbara), Chun Li Bai (Chinese Academy of Sciences, China), and David Mack (Indiana University) for valuable suggestions. This work was in part supported by the Whitaker Foundation and Sumitomo Electric Industries. Received for review November 3, 1999. Accepted April 27, 2000. AC991263I
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