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.

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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><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 &amp;amp; blocks for 0.5 mL tubes</td><td>Carl Roth GmbH</td><td>Y264.1 &amp;amp; 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 &amp;mu;m capsule filter</td><td>Unity Lab Services</td><td>N/A</td><td>water deionization and filter unit</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</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 0.5 mL 50k Ultracell</td><td>Millipore Ireland Ltd.</td><td>UFC505096</td><td>centrifuge filters</td></tr><tr><td>NucleoSpin Extract II&amp;nbsp;</td><td>&amp;nbsp;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 &amp;mu;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, MgCl&lt;sub&gt;2&lt;/sub&gt;, 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</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&amp;nbsp; 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&amp;nbsp;</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...

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

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 ...

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DNA Repair and Recombination

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9.4K Views.  University of Würzburg. 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.

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

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Cancer Research

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Biochemistry

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Visualization of DNA Repair Proteins Interaction by Immunofluorescence

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA insults. Genotoxic agents can damage the DNA backbone, break it, or modify the chemical nature of individual bases. Following DNA insult, DNA damage response (DDR) pathways are activated and proteins involved in the repair are recruited. A plethora of factors are involved in sensing the type of damage and activating the appropriate repair response. Failure to correctly activate and recruit DDR factors can lead to genomic instability, which underlies many human pathologies including cancer. Studies of DDR proteins can provide insights into genotoxic drug response and cellular mechanisms of drug resistance.
There are two major ways of visualizing&#160;proteins in vivo: direct observation, by tagging the protein of interest with a fluorescent protein and following it by live imaging, or indirect immunofluorescence on fixed samples. While visualization of fluorescently tagged proteins allows precise monitoring over time, direct tagging in N- or C-terminus can interfere with the protein localization or function. Observation of proteins in their unmodified, endogenous version is preferred. When DNA repair proteins are recruited to the DNA insult, their concentration increases locally and they form groups, or &#8220;foci&#8221;, that can be visualized by indirect immunofluorescence using specific antibodies.
Although detection of protein foci does not provide a definitive proof of direct interaction, co-localization of proteins in cells indicates that they regroup to the site of damage and can inform of the sequence of events required for complex formation. Careful analysis of foci spatial overlap in cells expressing wild type or mutant versions of a protein can provide precious clues on functional domains important for DNA repair function. Last, co-localization of proteins indicates possible direct interactions that can be verified by co-immunoprecipitation in cells, or direct pulldown using purified proteins.

Following DNA damage, human cells activate essential repair pathways to restore the integrity of their genome. Here, we describe the method of indirect immunofluorescence as a means to detect DNA repair proteins, analyze their spatial and temporal recruitment, and help interrogate protein-protein interaction at the sites of DNA damage.

Mammalian cells are constantly exposed to chemicals, radiations, and naturally occurring metabolic by-products, which create specific types of DNA ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

Nucleotide Excision Repair

<h4>Overview</h4>

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged DNA can be detected and repaired in a process called nucleotide excision repair (NER). NER employs a set of specialized proteins that first scan DNA to detect a damaged region. Next, NER proteins separate the strands and excise the damaged area. Finally, they coordinate the replacement with new, matching nucleotides.

<h4>DNA distortion and damage</h4>

Cells are regularly exposed to mutagens&mdash;factors in the environment which can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes to DNA. These include bends or kinks in the structure which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations which in turn can result in cancer or disease depending on which sequences are disrupted.

<h4>Identification and repair of damaged regions</h4>

Nucleotide excision repair relies on specific protein complexes to recognize damaged regions of DNA and flag them for removal and repair. In prokaryotes, the process involves three proteins&mdash;UvrA, UvrB, and UvrC. The first two proteins work together as a complex, traveling along the DNA strands to detect any physical aberrations.

Once identified, the strands at the damaged location are separated, and endonuclease enzymes such as UvrC cut and excise the affected region. DNA polymerase fills the gap with new nucleotides, and then the DNA ligase enzyme seals the edges between the new and old DNA.

<h4>Mutations in NER have severe consequences</h4>

In prokaryotes, the NER complex consists of the three Uvr proteins, but in eukaryotes, more than a dozen proteins operate to regulate DNA repair. In humans, mutations in the NER pathway can cause diseases such as xeroderma pigmentosum (XP), which is associated with a 2000-fold increase in the incidence of skin cancer. Individuals suffering from XP are highly sensitive to UV exposure and can develop severe skin burns after just a few minutes of exposure to sunlight. Additionally, XP patients can show signs of premature aging and often develop neurological abnormalities. Without a properly working repair mechanism, DNA damage can accumulate and lead to abnormal cell death or potentially cancerous tumors.

DNA Damage and Nucleotide Excision Repair

Overview

Exposure to mutagens can damage DNA and result in bulky lesions that distort the double-helix structure or impede proper transcription. Damaged ...