PUC19N, CPD-containing oligonucleotides, and p44 were kindly provided by Samuel Wilson, Korbinian Heil and Thomas Carell, and Gudrun Michels and Caroline Kisker, respectively. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) FZ82 and TE-671/4 to IT.

1. Sample Preparation
<ol>
	<li>Preparation of DNA substrates11
	<ol>
		<li>Generating a ssDNA gap in the plasmid
		<ol>
			<li>Completely digest a sample of the plasmid (here: modified pUC19, pUC19N) in a reaction tube with an appropriate nickase (here: Nt.BstNBI) followed by enzyme heat inactivation, using conditions according to the manufacturer&#39;s protocol (see Figure 1 for a schematic presentation). Start with ~50 &#181;L and ~500 nM plasmid for a sufficient yield.</li>
...

<ol>
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USA. 100 (25), 14822-14827 (2003).</li><li>Tessmer, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+MutS+searching+for+DNA+mismatches+and+signaling+repair.">Mechanism of MutS searching for DNA mismatches and signaling repair.</a> J. Biol. Chem. 283 (52), 36646-36654 (2008).</li><li>Wagner, K., Moolenaar, G., van Noort, J., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+analysis+reveals+two+separate+DNA-binding+domains+in+the+Escherichia+coli+UvrA+dimer.">Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.</a> Nucleic Acids Res. 37 (6), 1962-1972 (2009).</li><li>Buechner, C. 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Opin. Struct. Biol. 22, 88-93 (2012).</li><li>Buechner, C. N., Tessmer, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+substrate+preparation+for+atomic+force+microscopy+studies+of+protein-DNA+interactions.">DNA substrate preparation for atomic force microscopy studies of protein-DNA interactions.</a> J. Mol. Recognit. 26 (12), 605-617 (2013).</li><li>Sun, Z., Tan, H. Y., Bianco, P. R., Lyubchenko, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+RecG+Helicase+at+the+DNA+Replication+Fork+by+SSB+Protein.">Remodeling of RecG Helicase at the DNA Replication Fork by SSB Protein.</a> Sci. Rep. 5, 9625 (2015).</li><li>Billingsley, D. J., Bonass, W. A., Crampton, N., Kirkham, J., Thomson, N. 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Biol. 9 (2), 021001 (2012).</li><li>Maurer, S., Fritz, J., Muskhelishvili, G., Travers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+and+an+activator+form+discrete+subcomplexes+in+a+transcription+initiation+complex.">RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex.</a> EMBO J. 25 (16), 3784-3790 (2006).</li><li>Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. 1, (2012).</li><li>Theis, K., Chen, P. J., Skorvaga, M., Van Houten, B., Kisker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+UvrB,+a+DNA+helicase+adapted+for+nucleotide+excision+repair.">Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.</a> EMBO J. 18 (24), 6899-6907 (1999).</li><li>Kuper, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+TFIIH,+XPD+helicase+is+exclusively+devoted+to+DNA+repair.">In TFIIH, XPD helicase is exclusively devoted to DNA repair.</a> PLoS Biol. 12 (9), e1001954 (2014).</li><li>Shlyakhtenko, L. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silatrane-based+surface+chemistry+for+immobilization+of+DNA,+protein-DNA+complexes+and+other+biological+materials.">Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.</a> Ultramicroscopy. 97, 279-287 (2003).</li><li>Roth, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPB+helicase+regulates+DNA+incision+by+the+Thermoplasma+acidophilum+endonuclease+Bax1.">XPB helicase regulates DNA incision by the Thermoplasma acidophilum endonuclease Bax1.</a> DNA Repair. 11 (3), 286-293 (2012).</li><li>Ratcliff, G. C., Erie, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Single-Molecule+Study+to+Determine+Protein-Protein+Association+Constants.">A Novel Single-Molecule Study to Determine Protein-Protein Association Constants.</a> J. Am. Chem. Soc. 123 (24), 5632-5635 (2001).</li><li>Yang, Y., Sass, L. E., Du, C., Hsieh, P., Erie, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+protein-DNA+binding+constants+and+specificities+from+statistical+analyses+of+single+molecules:+MutS-DNA+interactions.">Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.</a> Nucleic Acids Res. 33 (13), 4322-4334 (2005).</li><li>Caron, P. R., Grossman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+a+cryptic+ATPase+activity+of+UvrB+and+its+proteolysis+product,+UvrB*+in+DNA+repair.">Involvement of a cryptic ATPase activity of UvrB and its proteolysis product, UvrB* in DNA repair.</a> Nucleic Acids Res. 16 (22), 10891-10902 (1988).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UvrB+domain+4,+an+autoinhibitory+gate+for+regulation+of+DNA+binding+and+ATPase+activity.">UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity.</a> J. Biol. Chem. 281 (22), 15227-15237 (2006).</li><li>Chammas, O., Billingsley, D. J., Bonass, W. A., Thomson, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+loops+as+fiducial+markers+for+exploring+DNA-protein+interactions+in+single+molecule+imaging.">Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging.</a> Methods. 60 (2), 122-130 (2013).</li><li>Truglio, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+DNA+recognition+and+processing+by+UvrB.">Structural basis for DNA recognition and processing by UvrB.</a> Nat. Struct. Mol. Biol. 13 (4), 360-364 (2006).</li><li>Kuper, J., Wolski, S. C., Michels, G., Kisker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+structural+studies+of+the+nucleotide+excision+repair+helicase+XPD+suggest+a+polarity+for+DNA+translocation.">Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.</a> EMBO J. 31 (2), 494-502 (2012).</li><li>Verhoeven, E. E., Wyman, C., Moolenaar, G. F., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+presence+of+two+UvrB+subunits+in+the+UvrAB+complex+ensures+damage+detection+in+both+DNA+strands.">The presence of two UvrB subunits in the UvrAB complex ensures damage detection in both DNA strands.</a> EMBO J. 21 (15), 4196-4205 (2002).</li><li>Verhoeven, E. E., Wyman, C., Moolenaar, G. F., Hoeijmakers, J. H., Goosen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+nucleotide+excision+repair+complexes:+DNA+is+wrapped+by+UvrB+before+and+after+damage+recognition.">Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition.</a> EMBO J. 20 (3), 601-611 (2001).</li><li>Moolenaar, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+ATP+Binding+and+Hydrolysis+by+UvrB+during+Nucleotide+Excision+Repair.">The Role of ATP Binding and Hydrolysis by UvrB during Nucleotide Excision Repair.</a> J. Biol. Chem. 275, 8044-8050 (2000).</li></ol>

The authors have nothing to disclose.

AFM statistical analyses of binding positions of proteins on long DNA fragments that contain specific target sites can reveal interesting details on the particular strategies employed by the protein to recognize these sites2,3,4,5,6. To interpret the resulting position distributions, the positions of the targets in the DNA need to be precisely known. This is a...

Atomic force microscopy (AFM) is a powerful technique for the analysis of protein-DNA interactions1,2,3,4,5,6,7,8,9. It requires only low amounts of sample material to directly visualize heterogeneous samples with a resolution at the single molecule level. Heterogeneity can result from different conformational or oligomeric states of a protein. In particular, in the context of protein-DNA samples, protein complexes can display different stoichiometries and/or conformations induced by DNA binding in general or binding to a specific target site within DNA. Heterogeneous samples may also contain two (or more) different kinds of proteins, and different protein complex forms (e.g., consisting of only one type of protein versus heteromeric complexes) may interact differently with DNA. The studies discussed here exploit AFM imaging in air on static, dried samples of DNA repair proteins bound to long (~900 base pairs, bp) DNA fragments that contain a lesion, which represents a target of these proteins. The high, molecular resolution of AFM allows the distinction between different types of protein complexes and to determine the binding positions of the proteins on the DNA fragments. Importantly, the lesions are introduced into the DNA substrates at well-defined positions. Because the position of the lesion site in the DNA is known, the distributions of proteins bound on DNA provide insight into (different) lesion recognition properties of the (different) protein complexes, e.g., how well they recognize a particular type of lesion (compared to non-damaged DNA)2,3,4,5,6. Their positions on the DNA also allow the distinction between protein complexes bound specifically at the lesions and complexes bound nonspecifically elsewhere on the DNA. Separate characterization of these different complex types (complexes bound specifically at the lesion versus nonspecific complexes) can reveal potential conformational changes in the complexes induced upon target site identification.
The DNA repair proteins focused on here are helicases that are responsible for lesion recognition in the nucleotide excision repair (NER) pathway. In bacteria, NER is achieved by the proteins UvrA, UvrB, and UvrC. UvrA is responsible for initial lesion sensing in a UvaA2/UvrB2 DNA-scanning complex. Upon lesion verification by UvrB this complex converts to monomeric UvrB bound at the lesion site and this specific complex can then recruit the prokaryotic NER endonuclease UvrC. UvrC excises a short (12-13 nt) stretch of single stranded DNA (ssDNA) containing the lesion. The missing stretch is then refilled by DNA polymerase. Finally, DNA ligase seals the newly synthesized stretch with the original DNA9,10. In eukaryotes, most proteins of the NER cascade are part of the large, multimeric transcription factor II H (TFIIH) complex. After initial lesion sensing via the trimeric CEN2-XPC-HR23B complex, TFIIH is recruited to the DNA target site. When XPD within the complex verifies the presence of an NER target lesion, the eukaryotic NER endonucleases XPG and XPF are recruited to excise a short (24-32 nt) stretch of ssDNA containing the lesion9,10. Here, specifically, the helicases UvrB and XPD from prokaryotic and eukaryotic NER, respectively, were studied. These helicases require an unpaired region in the DNA (a DNA bubble) to thread onto one of the two DNA single strands and subsequently translocate along this strand fueled by ATP hydrolysis. In addition to the DNA lesions, a DNA bubble was hence introduced in the substrates that functions as loading site for the proteins.
The procedure for preparation of specific lesion DNA substrates has been described previously11. It requires a circular DNA construct (plasmid) with two closely spaced restriction sites for a nickase. In the context of this study, the plasmid pUC19N (2729 bp) was used (created by S. Wilson&#39;s laboratory, NIEHS). This plasmid contains three closely spaced restriction sites for the nickase Nt.BstNBI that frame a 48 nucleotide (nt) stretch. After incubation with the nickase, the stretch of ssDNA between these sites can be removed and replaced by an oligonucleotide containing any target feature. After each step, complete enzymatic digestion is tested via agarose gel electrophoresis. Nicked circular DNA can be distinguished due to its lower electrophoretic mobility compared to the original supercoiled plasmid. Gapping of the DNA and replacement of the removed stretch by the specific substrate oligonucleotide can be evaluated via digestion with a restriction enzyme which incises the substrate exclusively within the region between the nicks. Linearization of the circular plasmid by the enzyme will hence be suppressed for the gapped DNA and restored after insertion of the specific oligonucleotide. Finally, two endonuclease restriction sites (ideally single cutters) allow for the generation of a linear DNA substrate, with length as desired and with the specific target site at a defined position as well as a DNA bubble at a distance from the lesion either in 5&#39; or 3&#39; direction.
Recognition of the lesions by the NER helicases can be investigated via AFM imaging. Stalled DNA translocation of the helicases at the lesion site is visible as a peak in the protein position distribution on the DNA and indicates lesion recognition. Because DNA translocation of these helicases is furthermore directional, with 5&#39;-to-3&#39; polarity, the dependence of lesion recognition on the position of the loading site (DNA bubble upstream or downstream from the lesion) also indicates whether the lesion is preferentially recognized on the translocated or on the opposite, non-translocated ssDNA strand5,9. In the following sections, the methods used will be introduced and major findings from these experiments will be briefly discussed. Importantly, analogous to the exemplary work on DNA repair shown here, AFM imaging can be applied to the study of different DNA interacting systems, such as DNA replication or transcription8,12,13,14.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Molecular Force Probe (MFP) 3D</td>
			<td>Asylum Research</td>
			<td>N/A</td>
			<td>atomic force microscope (AFM)</td>
		</tr>
		<tr>
			<td>Precision 390</td>
			<td>DELL</td>
			<td>N/A</td>
			<td>computer</td>
		</tr>
		<tr>
			<td>ThermoMixer and 1.5 ml block</td>
			<td>Eppendorf</td>
			<td>5382000015</td>
			<td>heat block for DNA preparation</td>
		</tr>
		<tr>
			<td>Rotilabo Block-Heater H 250 &#38; blocks for 0.5 ml tubes</td>
			<td>Carl Roth GmbH</td>
			<td>Y264.1 &#38; Y267.1</td>
			<td>heat block for protein-DNA incubations</td>
		</tr>
		<tr>
			<td>Mini-Sub Cell GT</td>
			<td>Bio-Rad Laboratories GmbH</td>
			<td>1704467</td>
			<td>electrophoresis chamber with gel caster and power supply</td>
		</tr>
		<tr>
			<td>Power Pac Basic</td>
			<td>Bio-Rad Laboratories GmbH</td>
			<td>1645050</td>
			<td>electrophoresis power supply</td>
		</tr>
		<tr>
			<td>Centifuge 5415 D with rotor</td>
			<td>Eppendorf</td>
			<td>2262120-3</td>
			<td>table centrifuge</td>
		</tr>
		<tr>
			<td>Ultra-Lum electronic UV transillumonator MEB-15</td>
			<td>Ultralum</td>
			<td>900-1322-02</td>
			<td>UV irradiation table</td>
		</tr>
		<tr>
			<td>NanoDrop ND-1000</td>
			<td>VWR International / PEQLAB Biotechnologie GmbH</td>
			<td>N/A</td>
			<td>UV spectrophotometer</td>
		</tr>
		<tr>
			<td>TKAX-CAD with 0.2 &#956;m capsule filter</td>
			<td>Unity Lab Services</td>
			<td>N/A</td>
			<td>water deionization and filter unit</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MFP software on Igor Pro</td>
			<td>Asylum Research</td>
			<td>N/A</td>
			<td>AFM software</td>
		</tr>
		<tr>
			<td>ImageJ (open source Java image processing)</td>
			<td>NIH Image</td>
			<td>N/A</td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Excel (Microsoft Office)</td>
			<td>Microsoft Corporation</td>
			<td>N/A</td>
			<td>data analysis software</td>
		</tr>
		<tr>
			<td>Origin9 / Origin2016</td>
			<td>OriginLab Corporation</td>
			<td>N/A</td>
			<td>statistical data analysis and graphing software</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OMCL-AC240TS</td>
			<td>Olympus</td>
			<td>OMCL-AC240TS</td>
			<td>AFM cantilevers</td>
		</tr>
		<tr>
			<td>grade V-5 muscovite</td>
			<td>SPI Supplies</td>
			<td>1805</td>
			<td>mica sheets</td>
		</tr>
		<tr>
			<td>Amicon Ultra 0.5ml 50k Ultracell</td>
			<td>Millipore Ireland Ltd.</td>
			<td>UFC505096</td>
			<td>centrifuge filters</td>
		</tr>
		<tr>
			<td>NucleoSpin Extract II&#160;</td>
			<td>&#160;Macherey-Nagel GmbH</td>
			<td>740 609.250</td>
			<td>Agarose gel extraction kit</td>
		</tr>
		<tr>
			<td>Rotilabo cellulose paper type 111A</td>
			<td>Carl Roth GmbH</td>
			<td>AP59.1</td>
			<td>AFM deposition blotting paper</td>
		</tr>
		<tr>
			<td>Anatop 25 (0.02 &#956;m)</td>
			<td>Whatman GmbH</td>
			<td>6809-2102</td>
			<td>syringe filter</td>
		</tr>
		<tr>
			<td>SSpI, BspQI</td>
			<td>New England Biolabs (NEB)</td>
			<td>R0132, R0712</td>
			<td>restriction enzymes for DNA substrate preparation</td>
		</tr>
		<tr>
			<td>XhoI, BglII</td>
			<td></td>
			<td>R0146, R0144</td>
			<td>restriction enzymes for DNA preparation controls</td>
		</tr>
		<tr>
			<td>nicking restriction enzyme Nt.BstNBI</td>
			<td>New England Biolabs (NEB)</td>
			<td>R0607</td>
			<td>nickase</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>New England Biolabs (NEB)</td>
			<td>M0202S</td>
			<td>Ligase</td>
		</tr>
		<tr>
			<td>Tris, HEPES</td>
			<td>Carl Roth GmbH</td>
			<td>4855, 9105</td>
			<td>buffer chemicals</td>
		</tr>
		<tr>
			<td>NaCl, MgCl2, KCl, MgAcetate</td>
			<td>Carl Roth GmbH</td>
			<td>3957, HN03, HN02, P026</td>
			<td>salt chemicals</td>
		</tr>
		<tr>
			<td>NaAc</td>
			<td>Sigma-Aldrich Chemie GmbH</td>
			<td>32318</td>
			<td>salt chemicals</td>
		</tr>
		<tr>
			<td>DTT, TCEP, EDTA</td>
			<td></td>
			<td>6908, HN95, 8040</td>
			<td>chemicals/reagents</td>
		</tr>
		<tr>
			<td>agarose, acetic acid, HCl</td>
			<td>Carl Roth GmbH</td>
			<td>2267, 3738, K025</td>
			<td>reagents</td>
		</tr>
		<tr>
			<td>ATP&#404;S</td>
			<td>Jena Bioscience</td>
			<td>NU-406</td>
			<td>nucleotides</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Carl Roth GmbH</td>
			<td>K054</td>
			<td>nucleotides</td>
		</tr>
		<tr>
			<td>oligonucleotide #1 in Table 1</td>
			<td>Biomers</td>
			<td>custom</td>
			<td>complementary DNA oligonucleotide</td>
		</tr>
		<tr>
			<td>oligonucleotides #2, #3, and #6 in Table 1</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>custom</td>
			<td>fluorescein containing oligonucleotides</td>
		</tr>
		<tr>
			<td>oligonucleotides #4&#160; and #5 in Table 1</td>
			<td>private (available from e.g. TriLink or GlenResearch)</td>
			<td></td>
			<td>CPD containing oligonucleotides</td>
		</tr>
		<tr>
			<td>SafeSeal reaction tube 0.5 ml and 1.5 ml</td>
			<td>Sarstedt</td>
			<td>72.704 and 72.706</td>
			<td>incubation tubes</td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb</td>
			<td>Thermo Scientific</td>
			<td>SM0311</td>
			<td>DNA ladder</td>
		</tr>
		<tr>
			<td>6x concentrate gel loading dye purple</td>
			<td>New England Biolabs (NEB)</td>
			<td>51406</td>
			<td>DNA loading dye</td>
		</tr>
		<tr>
			<td>Midori Green&#160;</td>
			<td>Nippon Genetics Europe GmbH</td>
			<td>999MG28055</td>
			<td>DNA stain</td>
		</tr>
	</tbody>
</table>

atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different molecules and molecular assemblies in a heterogeneous sample. In the particular context of DNA interacting protein systems, different protein complex forms and their corresponding binding positions on target sites containing DNA fragments can thus be distinguished. Here, an application of AFM to the study of DNA lesion recognition in the prokaryotic and eukaryotic nucleotide excision DNA repair (NER) systems is presented. The procedures of DNA and protein sample preparations are described and experimental as well as analytical details of the experiments are provided. The data allow important conclusions on the strategies by which target site verification may be achieved by the NER proteins. Interestingly, they indicate different approaches of lesion recognition and identification for the eukaryotic NER system, depending on the type of lesion. Furthermore, distinct structural properties of the two different helicases involved in prokaryotic and eukaryotic NER result in and explain the different strategies observed for these two systems. Importantly, these experimental and analytical approaches can be applied not only to the study of DNA repair but also very similarly to other DNA interacting protein systems such as those involved in replication or transcription processes.

Distinguishing between different complex types based on protein complex volumes
The helicase activity of the prokaryotic NER helicase UvrB is stimulated by DNA binding22,23. UvrB requires an unpaired region in the DNA (a DNA bubble) in order to load correctly onto one of the two single ssDNA strands. In vivo, this DNA structure is provided by DNA interactions throu...

Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Here, the study of different DNA lesion recognition approaches via single molecule AFM imaging is demonstrated with the nucleotide excision repair system as an example. The procedures of DNA and protein sample preparations and experimental as well as analytical details for the AFM experiments are described.

AFM imaging is a powerful technique for the study of protein-DNA interactions. This single molecule method allows the simultaneous resolution of different ...

The overall goal of this atomic force microscopy experiment is to resolve interactions of DNA repair proteins with damage sites in DNA and to distinguish between different DNA interactions by various protein complexes. This method can help answer key questions in the DNA repair field, such as how a particular DNA repair system achieves the recognition of its target lesions in the DNA. The main advantage of this technique is that we can directly see and analyze the different types of complexes that are involved in the DNA lesion recognition process.Demonstrating the procedure will be Nicolas Wirth, who used this approach during his bachelor research. To begin, prepare a suitable DNA substrate by generating a single-stranded DNA gap in a plasmid. Refill the gap with modified, single-stranded DNA oligonucleotides, and finally, linearize the plasma DNA using appropriate restriction enzymes.For these experiments, the double-stranded DNA substrates are about 1, 000 base pairs long and contain a DNA lesion as well as an unpaired region called a DNA bubble for protein loading onto the DNA. Next, prepare a protein DNA sample for AFM analysis by first pipetting 10x Reaction Buffer and water into a microreaction tube. Then, add the DNA and proteins under investigation.For example, here, 100 nanomolar of the DNA sample and one micromolar or each of the eukaryotic nucleotide excision repair proteins XPD and p44. Then, incubate the reaction for 30 minutes at 37 degrees Celsius in a heat block. Following the incubation, briefly spin down the sample.Next, prepare a mica substrate to place the sample onto. Use a scalpel to cut a one-centimeter by one-centimeter piece of mica from a larger strip. Then, use a piece of adhesive tape to remove the top layers of the mica to reveal a clean, flat, anatomically smooth substrate surface.Dilate the protein DNA sample fifty to a hundredfold in AFM deposition buffer, and immediately deposit 20 microliters of the diluted sample onto the mica surface. The dilution factor depends on the sample concentration. Immediately after depositing the sample on the mica surface, rinse the sample three to four times with a few milliliters of filtered, deionized water and blot off excess liquid.Use a gentle stream of nitrogen to blow dry the sample. Finally, use adhesive tape to fix the sample at its edges onto a microscope slide. Place the sample in the middle of the AFM stage and use magnetic pads to fix the microscope slide on the stage.Then, insert the AFM tip into the tip holder. Fix the tip in the holder under a clamp by tightening the screw so that it is finger tight. Once secured, enter the tip holder into the AFM measurement head.Use the positioning screws to move the sample in a central position. Place the AFM measurement head on top of the sample, making sure the head stands stably with its legs inside the stage indentations. Next, align the AFM laser on the back of the cantilever for optimal signal strength.Watch the reflection signal in the AFM video window to crudely position the laser. For this AFM model, turn the wheels of the right side and back of the AFM measurement head to adjust the X and Y positions of the AFM laser in order to direct it centrally onto the end of the cantilever. Then, optimize the detector sum signal by fine-tuning the laser position with the two wheels.Direct the AFM laser reflection onto the detector center by zeroing the different signal from the top and bottom diodes of the detector array. For this step, turn the screw on the left-side of the measurement head for this AFM model. Next, determine the cantilever residence frequency by going into the master panel tune window in the software.Choose an amplitude corresponding to a one-volt input for the Piezo that drives the cantilever oscillation and then set the oscillation frequency to negative 5%slightly lower than the residence frequency. Now perform an auto frequency tune and zero the phase of the oscillation. Leave the system to reach thermal equilibrium for about one hour before you start imaging, in order to avoid drift.When you are ready to start imaging, in the software main panel, set a protecting set point and prepare to approach the tip to the sample surface by selecting engage. Lower the front wheel of the AFM head manually until the protective setting, or set point, is reached. Then, carefully lower the front wheel further until the Z sensor signal is fully extended to the left.Activate the vibration oscillation table and close the acoustic isolation enclosure. Next, lower the set point in the AFM software using the hamster wheel to fine engage the AFM tip with the sample. To perform tapping mode imaging in the repulsive regime, lower the set point until the phase signal becomes slightly lower than the free level phase.Then, begin scanning the sample. Use the scan speed of 2.5 micrometers per second in image surface areas of four micrometer by four micrometer or eight micrometer by eight micrometer squares with pixel resolutions of 2, 048 or 4, 096, respectively;and press the Do Scan button. Preselect relevant protein DNA complexes by direct visual inspection of the images in the AFM software.For XPD samples, possible complexes include XPD p44 DNA, as well as XPD DNA and p44 DNA with distinctly different sizes due to the different molecular masses of XPD and p44. Measure volumes of individual protein peaks on the DNA by using the section tool in the analysis window to measure the height and diameter of the protein peak sections with the section window cursors. Use these values to determine the volume of the peaks using simple mathematical models to verify the complex type.Finally, calibrate your AFM system with a range of proteins with known molecular weights to translate these volumes into approximate molecular weights of proteins and protein complexes. In order to determine additional features, such as DNA fragment lengths, the positions of protein complexes on the DNA and their target-site specificities or DNA band angles, see the accompanying text protocol. Shown here is an AFM image with three different types of DNA-bound complexes that can be distinguished based on their different sizes.Class one complexes are consistent with only the helicase activating co-factor p44, class two complexes consist of only the XPD protein, and class three complexes consist of both XPD and p44 as legion recognition by XPD requires the activation of its helicase activity by p44, and only the class three complexes show lesion recognition. In order to identify different lesion recognition strategies, the locations of proteins on the DNA substrates were measured for protein loading at a DNA bubble in either the five prime or three prime directions from a DNA lesion shown in red for prokaryotic and eukaryotic NER helicases. In the protein position distributions, DNA lesion recognition can be seen as a peak due to enhanced binding at the DNA lesion position.The prokaryotic NER helicase UVRB and its eukaryotic counterpart, XPD, show different strategies of CPD lesion recognition that indicate different DNA strand preferences. Specifically, CPD lesions are recognized on the translocated strand by UVRB, but preferentially on the opposite, non-translocated strand by XPD. Once mastered, this method can be done in a few hours or days including analysis.It's important to work with highly pure protein and DNA samples to be able to interpret the features in the AFM images and to carefully design the DNA substrate depending on the biological system and on the question you want to address. In addition, ensemble methods, such as gel electrophoresis space, electromobility shift, or DNA incision assays are, of course, highly complementary to these AFM experiments;and these methods can help address additional questions, such as on DNA binding affinities or DNA lesion excision activities. This technique is a relatively fast and simple approach to directly visualize protein DNA complexes, and these complexes give us a good picture of the processes that occur in a biological system.Of course, this is not only useful in understanding DNA repair processes, but it can also be applied to other protein DNA systems. After watching this video, you should have a pretty good idea of how to prepare a sample on your favorite protein DNA system, how to image it by AFM, and of how to extract useful parameters for an interpretation of your data.

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9.3K visualizações.  University of Würzburg. The overall goal of this atomic force microscopy experiment is to resolve interactions of DNA repair proteins with damage sites in DNA and to distinguish between different DNA interactions by various protein complexes. This method can help answer key questions in the DNA repair field, such as how a particular DNA repair system achieves the recognition of its target lesions in the DNA. The main advantage of this technique is that we can directly see and analyze the different types of complexes...