Name: atomic force microscopy investigations of dna lesion recognition in nucleotide excision repair
Uploaded: 2017-05-24T14:00:00
Description: Watch this Scientific Journal Video about Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair at JoVE.com

PUC19N, CPD- 함유 올리고 뉴클레오타이드 및 p44는 Samuel Wilson, Korbinian Heil 및 Thomas Carell, Gudrun Michels 및 Caroline Kisker에 의해 각각 제공되었다. 이 연구는 도이체 Forschungsgemeinschaft (DFG) FZ82 및 TE-671 / 4에서 IT 보조금으로 지원되었습니다.

1. 샘플 준비 <ol><li> DNA 기판 준비 11 <ol><li> 플라스미드에서 ssDNA 갭 생성 <ol><li> 적절한 니카아제 (여기 Nt.BstNBI)와 효소 열 불 활성화와 반응 튜브에서 플라스미드 (여기에 : 수정 된 pUC19, pUC19N)의 샘플을 완전히 소화기 제조 업체의 프로토콜에 따라 조건을 사용하여 소화 (그림 1 참조 설계도 표시). 충분한 수율을 얻기 위해 ~ 50 μL와 ~ 500 nM 플라스미드로 시작하십시?...

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특정 표적 부위를 포함하는 긴 DNA 단편에 대한 단백질의 결합 위치에 대한 AFM 통계 분석은 단백질이 이들 부위 2 , 3 , 4 , 5 , 6 을 인식하기 위해 사용하는 특정 전략에 대한 흥미로운 세부 사항을 나타낼 수있다. 결과적인 위치 분포를 해석하기 위해, DNA 내 표적의 위치를 ​?...

원자 힘 현미경 (AFM)은 단백질 -DNA 상호 작용 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 의 분석을위한 강력한 기술입니다. 단일 분자 수준의 분해능으로 이종 샘플을 직접 시각화하기 위해서는 적은 양의 시료가 필요합니다. 이질성은 단백질의 상이한 형태 또는 올리고머 상태에 기인 할 수있다. 특히, 단백질 -DNA 샘플과 관련하여, 단백질 복합체는 일반적으로 DNA 결합 또는 DNA 내의 특정 표적 부위에 결합함으로써 유도 된 상이한 화학 양론 및 / 또는 형태를 나타낼 수있다. 이종 샘플은 또한 2 종 (또는 그 이상)의 상이한 종류의 단백질 및 상이한 단백질 복합체형태 ( 예를 들어 , 하나의 유형의 단백질 대 헤테로 메릭 복합체로 이루어진)는 DNA와 다르게 상호 작용할 수있다. 여기서 논의 된 연구는 공기 중의 AFM 이미징을 이용하여이 단백질의 표적을 나타내는 병변을 포함하는 긴 (~ 900 염기쌍, bp) DNA 단편에 결합 된 DNA 복구 단백질의 정적 건조 샘플을 이용한다. AFM의 분자 분해능이 높기 때문에 여러 종류의 단백질 복합체를 구별 할 수 있으며 DNA 조각에 결합하는 단백질의 결합 위치를 결정할 수 있습니다. 중요하게도, 병변은 잘 정의 된 위치에서 DNA 기질에 도입됩니다. DNA에 병변 부위의 위치가 알려지기 때문에 DNA에 결합 된 단백질의 분포는 (상이한) 단백질 복합체의 (상이한) 병변 인식 특성, 예를 들어 그들이 특정 유형의 병변을 얼마나 잘인지하는지에 대한 통찰력을 제공한다 손상되지 않은 DNA에) 2 , 3 , <supclass = &quot;xref&quot;&gt; 4 , 5 , 6 . DNA에 대한 그들의 위치는 또한 병변에서 특이 적으로 결합 된 단백질 복합체와 DNA의 다른 곳에서 비특이적으로 결합 된 복합체 사이의 구별을 허용한다. 이들 상이한 복합 형 (병변 및 비 특이성 복합체에서 특이 적으로 결합 된 복합체)의 분리 된 특성화는 표적 부위 확인시 유도 된 복합체에서의 잠재적 인 구조 변화를 나타낼 수있다. 여기에 초점을 맞춘 DNA 복구 단백질은 핵산 절삭 수선 (NER) 경로에서 병변 인식을 담당하는 헬리 카아 제입니다. 박테리아에서 NER은 단백질 UvrA, UvrB 및 UvrC에 의해 달성됩니다. UvrA는 UvaA 2 / UvrB 2 DNA 스캐닝 복합체에서 초기 병변 감지를 담당합니다. UvrB에 의한 병변 검증시,이 복합체는 병변 부위에서 결합 된 단량체 UvrB로 전환되고,이 특정 복합체는 p원핵 세포 NER 엔도 뉴 클레아 제 UvrC. UvrC는 병변이있는 짧은 (12-13nt) 스트레치의 단일 가닥 DNA (ssDNA)를 절단합니다. 누락 된 스트레치는 DNA 폴리머 라제에 의해 재충전됩니다. 마지막으로, DNA 리가 제가 원래 합성 된 DNA 9,10 으로 새로 합성 된 신축성을 봉인합니다. 진핵 생물에서 NER 캐스케이드의 대부분의 단백질은 대형의 다중 전사 인자 II H (TFIIH) 복합체의 일부입니다. 삼량 체 CEN2-XPC-HR23B 복합체 를 통한 초기 병변 감지 후, TFIIH는 DNA 표적 부위로 모집된다. 복합체 내의 XPD가 NER 표적 병변의 존재를 확인하면, 진핵 생물 NER 엔도 뉴 클레아 제인 XPG 및 XPF는 병변 9 , 10을 함유 한 짧은 (24-32 nt) ssDNA 스트레치를 모집하도록 모집된다. 여기서는 원핵 생물과 진핵 생물 NER의 헬리 케이즈 UvrB와 XPD를 연구했습니다. 이 헬리콥터에는DNA (DNA bubble)는 두 개의 DNA 단일 가닥 중 하나에 연결되고,이어서 ATP 가수 분해에 의해 연료 가닥을 따라 이동합니다. 따라서 DNA 병변 이외에 DNA 버블이 단백질의 로딩 위치로 기능하는 기질에 도입되었습니다. 특정 병변 DNA 기판의 준비 절차는 이전에 11에서 설명 했습니다. 그것은 니카아제를위한 2 개의 밀접하게 이격 된 제한 부위를 갖는 원형 DNA 구조체 (플라스미드)를 필요로한다. 이 연구의 맥락에서, 플라스미드 pUC19N (2729 BP)이 사용되었다 (S. Wilson의 실험실, NIEHS에 의해 만들어졌다). 이 플라스미드는 48 뉴클레오타이드 (nt) 스트레치를 구성하는 니카타아제 Nt.BstNBI를위한 3 개의 밀접하게 이격 된 제한 사이트를 포함한다. 니카아제로 배양 한 후, 이들 부위 사이의 ssDNA의 스트레치를 제거하고 임의의 표적 특징을 함유하는 올리고 뉴클레오타이드로 대체 할 수있다. 각 단계 후, 완전한 효소 소화를 아가 로즈 겔로 시험한다전기 영동. Nicked 원형 DNA는 원래 supercoiled 플라스미드에 비해 낮은 전기 영동 이동성으로 인해 구별하실 수 있습니다. DNA의 갭핑 및 제거 된 스트레치의 특정 기질 올리고 뉴클레오타이드에 의한 대체는 닉 사이의 영역 내에서만 기질을 절개하는 제한 효소로 분해함으로써 평가 될 수있다. 효소에 의한 원형 플라스미드의 선형화는 따라서 갭 DNA에 대해 억제되고 특정 올리고 뉴클레오타이드의 삽입 후에 회복 될 것이다. 마지막으로, 두 endonuclease 제한 사이트 (이상적으로 단일 커터) 원하는대로 길이와 정의 된 위치뿐만 아니라 병변에서 거리에있는 DNA 버블에서 특정 대상 사이트와 선형 DNA 기판의 생성을 허용 5 &#39;또는 3&#39;방향. NER 헬리 케이즈에 의한 병변의인지 는 AFM 영상 을 통해 조사 할 수 있습니다. t에서 헬리 카스의 DNA 전좌가 스톨 된그 병변 부위는 DNA상의 단백질 위치 분포의 첨단으로 볼 수 있으며 병변 인식을 나타낸다. 이 헬리 카제의 DNA 전좌가 5 &#39;에서 3&#39;극성으로 나아가 방향성을 갖기 때문에 로딩 위치 (병변의 상류 또는 하류의 DNA 버블)의 위치에 대한 병변 인식의 의존성도 병변이 우선적으로인지되는지 여부를 나타냅니다 (translocated) 또는 반대쪽으로, 비 - 전위 된 ssDNA 가닥 5 , 9 . 다음 섹션에서는 사용 된 방법을 소개하고이 실험의 주요 결과를 간단히 설명합니다. 중요한 것은 여기에 제시된 DNA 수리에 대한 모범 사례와 유사하게 AFM 이미징은 DNA 복제 또는 전사 8 , 12 , 13 , 14 와 같은 서로 다른 DNA 상호 작용 시스템의 연구에 적용될 수 있습니다 </sup&gt;.

<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.

단백질 복합체 부피를 기준으로 다른 복합 형을 구별 원핵 생물 NER 헬리 케이즈 UvrB의 헬리 케이즈 활성은 DNA 결합에 의해 자극된다 22 , 23 . UvrB는 두 개의 단일 ssDNA 가닥 중 하나에 올바르게로드하기 위해 DNA에 짝을 이루지 않은 영역 (DNA 버블)이 필요합니다. 생체 내 에서,이 DN...

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.

핵 포스 현미경 검사에 의한 DNA 절편 검사에서의 DNA 병변 인식 연구

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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