Atomic Force Microscopy Onderzoek naar DNA Lesion Recognition in Nucleotide Excision Repair

Summary

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.

Abstract

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.

Introduction

Atomische krachtmicroscopie (AFM) is een krachtige techniek voor de analyse van eiwit-DNA-interacties ^{1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9} . Het vereist slechts lage hoeveelheden monstermateriaal om heterogene monsters rechtstreeks te visualiseren met een resolutie op het molecuulniveau. Heterogeniteit kan resulteren uit verschillende conformatieve of oligomere toestanden....

Protocol

1. Voorbeeld Bereiding

1. Bereiding van DNA-substraten ¹¹
   1. Het genereren van een ssDNA-kloof in het plasmide
      1. Volledig verteer een monster van het plasmide (hier: gemodificeerd pUC19, pUC19N) in een reactiebuis met een gepaste nickase (hier: Nt.BstNBI) gevolgd door enzym-inactivatie onder gebruikmaking van condities volgens het protocol van de fabrikant (zie Figuur 1 voor een schematische presentatie). Begin met ~ 50 μL en ~ 500 nM plasmide voor een voldoende opbrengst.
      2. Verifieer plasmide nicking door agarosegel elektroforese op verdunde monsters (~ 20 nM) ¹⁵ . Draag ha....

Results

Onderscheid tussen verschillende complexe types op basis van eiwitcomplexvolumes

De helicase activiteit van het prokaryotische NER helicase UvrB wordt gestimuleerd door DNA-binding ^{22 , 23} . UvrB vereist een ongepaarde regio in het DNA (een DNA-bubble) om correct op een van de twee single ssDNA-strengen te laden. In vivo wordt deze DNA-structuur verschaft door DN.......

Discussion

AFM statistische analyses van bindende posities van eiwitten op lange DNA-fragmenten die specifieke doelstellingsplaatsen bevatten, kunnen interessante details aantonen over de specifieke strategieën die door het eiwit gebruikt worden om deze sites ^{2 , 3 , 4 , 5 , 6} te herkennen. Om de resulterende positieverdelingen te interpreteren, moeten de posities van d.......

Materials

List of materials used in this article

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