Moss Tom. DNA-protein interactions: principles and protocols

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616 Russman, Beigang, and Beato

crosslinking yield with the TWF method (14%) as compared with a single fs

UV pulse (4.8%).

4. Notes

1. The laser equipment used in the first experiments for TWF laser crosslinking is

rather complex and sophisticated. Molecular biologists interested in using this

method are best advised to contact a laser laboratory. However, the recent devel-

opment of compact, diode-laser-pumped, all-solid-state femtosecond lasers and

the use of new nonlinear materials for frequency conversion, like quasi-phase-

matched crystals, will eventually result in reliable easy-to-operate femtosecond

systems at a reasonable price. These systems are well-suited for routine applica-

tions of this new crosslinking method in molecular biology laboratories.

2. The irradiated samples should be as thin as possible to avoid dispersion effects,

as these cause a change of the temporal distance between the blue and the UV

pulses. The velocity of the positioning table should be precisely tunable. Changes

in velocity can then be used to vary the applied energy per square millimeter of

cell layer. This is an important parameter for increasing the in vivo crosslinking

yield. However, irradiation should be fast enough to avoid drying of the cells.

3. Many crosslinked protein-DNA complexes do not enter a standard SDS–protein

gel when solubilized in SDS–sample buffer without urea (3). Probably, urea pre-

vents the aggregation of crosslinked material.

4. The amount of full-length extension product is only an operational value,

depending on the irradiation conditions and on the length of the template. One

should keep in mind that this parameter is used to optimize the crosslink proce-

dure and does not represent a stringent measurement of DNA damage.

References

1. Rubmann, C., Truss, M., Fix, A., Naumer, C., Herrmann, T., Schmitt, J., et al.

(1997) Lasercrosslinking of progesterone receptor to DNA using tuneable nano-

second, picosecond and femtosecond laser pulses. Nucleic Acids Res. 25(12),

2478–2484.

2. Rubmann, C., Stollhof, J., Weib, C., Beigang, R., and Beato, M. (1998) Femto-

second two wavelength crosslinking of DNA–protein-complexes. Nucleic Acids

Res. 26(17), 3967–3971.

3. Ho, D. T., Sauvé, D. M., and Roberge, M. (1994) Detection and isolation of DNA-

binding proteins using single-pulse ultraviolet laser crosslinking. Anal. Biochem.

218, 248–254.

Appendix I 617

617

Appendix I

EMSA/Gel Shift Conditions:

Acrylamide:

Bisacrylamide Reference

Buffer Conditions Ratio Chapter

Tris-glycine: 50 mM Tris, 2,5 mM EDTA, 0,4 M 39:1 Ch 2

glycine

Tris-acetate: 6.7 mM Tris-HCl, pH 7.5, 3.3 mM — Ch 7

sodium acetate, 1 mM EDTA

0.5 × Tris-borate EDTA: 45 mM Tris, 45 mM 39:1 Ch 2

boric acid, 1 mM EDTA

1 × Tris-borate EDTA: 0.089 M Tris-borate, 37.5:1 Ch 32

pH 8.0, 1 mM EDTA

1 × Tris-borate CaCl

: 0.089 M Tris-borate, 37.5:1 Ch 32

pH 7.5 to 8.0 and up to 5 mM CaCl

Tris-HCl: 8 mM Tris-HCl (recycled) 39:1 Ch 4

HEPES-NaCl: 50 mM NaCl, 40 mM 30:0,8 Ch 37

HEPES-NaOH, pH 7.5 and 5% v/v glycerol. (=37.5:1)

Running buffer: 50 mM NaCl, 20 mM

HEPES-NaOH, pH 7.5

Appendix II 619

Reagent MW DNA Duplex Attack Comments Uses

1,10- 284 Ribose via minor groove. Does not attack Footprinting.

Z-form DNA

Phenanthroline- and more

Copper TAT>CG>TA weakly (35%)

attacks

A form DNA

as compared

to B form.

DEPC 162 G + A ¨ C, Out of plane attack DNA

unstacked bases on bases conformation,

base destack-

ing, bending.

Dimethyl Sulphate 126 N7-G via major groove Footprinting

(DMS) N3-A via minor groove Interference

DNase I 40k Independent attack on

Sequence specific Footprinting.

phosphodiester bonds DNA cleavage

from minor groove

face of DNA

Ethyl-nitroso-urea 117 60–65% Non-esterified Low sequence Interference

oxygen of specificity

phosphodiester (> T-O2

(minor groove) =G-O6

(major groove) > T-O4

(major groove) >> C-O2

(minor groove))

Exonuclease III 28k 3 terminal attack Extremities of “Footprinting”

DNA-protein

complexes

Continued...

Appendix II

DNA-Modification/Cleavage Reagents

619

620 Appendix II

Hydroxy-radical 17 Sugar backbone Little sequence Footprinting

preference Interference

Osmium tetroxide 254 Unstacked T Very specific for DNA

certain, as yet conformation

poorly defined

DNA

conformations

including

overwinding

Permanganate 119 T » G, C, A Out of plane attack DNA

on 5,6 double conformation,

bond of T melting, base

destacking,

bending.

Diffusible singlet 16 Unstaked/unwound DNA Little sequence DNA

oxygen bases preference in conformation

duplex DNA

(though as free

bases, G is by

far the prefered

target)

uranyl(VI) ion 268 Phosphates-photolysis Little sequence Footprinting

(UO

) dependence of both

macromol-

ecules and

drugs.

UV irradiation

—

TT >> CT, TC, or CC Products: Footprinting.

i) cyclobutane Note, protein

pyrimidine does not

dimer (CBD). prevent

ii) pyrimidine (6-4) access but

pyrimidone changes DNA

photoproduct reactivity

(6-4PP) locally.

Index 621

621

Index

AFM, see Atomic force microscopy

Aminopropyltrietoxy silane (APTES),

mica functionalization, see

Atomic force microscopy

1-Anilinonaphthalene-8-sulfonic acid

(ANS),

competition assay for DNA binding,

advantages over intrinsic

fluorescence, 265

binding curve generation, 270–272

materials, 267, 268, 273

preliminary testing, 266, 267,

269, 270, 273

principle, 265

resonance energy transfer, 273, 274

titration, 268, 269, 273

protein binding properties, 265, 266

ANS, see 1-Anilinonaphthalene-8-

sulfonic acid

APTES, see Aminopropyltrietoxy silane

Atomic force microscopy (AFM),

advantages and limitations, 569

functionalized mica substrates,

overview, 569, 570

preparation, 571, 575

imaging in air,

contact vs tapping modes,

569, 570

sample preparation,

droplet procedure, 569, 571,

572

immersion procedure, 569,

571, 572

imaging in solution,

advantages, 570

dried samples, 570, 572

in situ, 570, 571

materials for nucleoprotein imaging,

570

multivalent cation method for

sample preparation, 571

8-Azidoadenine,

DNA incorporation,

materials, 325, 326

nick translation, 328, 332

DNA–protein crosslinking,

filter binding assay, 330

gel electrophoresis analysis,

329, 330

photocrosslinking, 328, 329,

332, 333

photoaffinity labeling overview,

323, 324

synthesis,

characterization, 327, 328, 331,

332

materials, 325

protocol, 326, 327, 330, 331

radiolabeled compound, 330

Biacore, see Surface plasmon resonance

Calorimetry, see also Differential

scanning calorimetry; Isothermal

titration calorimetry,

DNA-binding protein applications, 512

622 Index

equilibrium, 513

thermodynamic parameters,

energetics of protein–DNA

interaction, 512, 513

overview, 511, 512

CD, see Circular dichroism

Circular dichroism (CD),

applications for DNA-binding

proteins, 504, 508

DNA change measurement upon

protein binding,

materials, 504, 505

titration, 505, 506, 508

wavelength selection, 504, 505

origin of signal, 503

principles, 503, 504

sensitivity, 504

Sox-5 HMG domain dissociation

constant determination for

DNA, 518

Crystallography, see Reconstitution,

protein–DNA complexes for

crystallization; Two-dimensional

crystallization

DEPC footprinting, see Diethyl

pyrocarbonate footprinting

Diethyl pyrocarbonate (DEPC)

footprinting,

advantages, 63, 64

applications, 64, 65

DNA modification,

detection of modified bases with

piperidine cleavage, 65, 66,

68, 69, 71

reaction mechanism, 64

in vitro experiments on linear

DNA fragments,

binding reaction, 68, 71

gel electrophoresis, 69, 71

modification reaction and

stopping, 68, 71

piperidine cleavage, 68, 69, 71

radiolabeling of probe, 68

materials, 67

Differential scanning calorimetry

(DSC), see also Calorimetry,

excess molar heat capacity, 516

intrinsic heat capacity, 516

partial molar heat capacity, 515, 516

principle, 515, 516

Sox-5 HMG domain interaction with

DNA,

concentration determinations,

complex, 528, 529

DNA, 527, 5286

protein, 528

correction of isothermal titration

calorimetry-derived

enthalpies, 525, 526

data acquisition, 524, 530, 531

data analysis, 525

instrumentation, 520

materials, 520, 521, 529

Dimethyl sulfate (DMS) footprinting,

combination with electrophoretic

mobility shift assay, 78, 79

ligation-mediated polymerase chain

reaction for in vivo

footprinting,

advantages and limitations, 183, 187

cleavage for DNA sequencing

products,

A reaction, 201

C reaction, 201, 202

G reaction, 201

overview, 200, 201

processing of samples, 202

reagants, 192

T+C reaction, 201

detectable DNA–protein

interactions, 183

dimethyl sulfate treatment, 193,

202, 203, 213

DNA polymerase selection, 191

Index 623

DNA purification,

DNA extraction, 200, 213

materials, 191, 192

nuclei isolation, 200

quantification, 200, 213

gel electrophoresis and

electroblotting,

blotting, 209, 214

electrophoresis, 208, 209

materials, 196, 197

hybridization,

digoxigenin-labeled probe,

197, 198, 210, 214, 215

materials, 197

radiolabeled probe, 197,

209, 214

ligation,

ligation reaction, 207

materials, 195

modified bases and conversion to

single-strand breaks, 181,

188, 193, 205

overview, 176, 177

polymerase chain reaction,

cycles, 207, 208

materials, 195, 196, 213

primer extension,

incubation conditions,

206, 207

materials, 194, 195, 212, 213

single-stranded hybridization

probe preparation,

amplification product

purification and

quantification, 198, 199,

211, 215

digoxigenin labeling, 199,

212, 215

length, 210, 215

materials, 198, 199, 213

polymerase chain reaction

amplification, 198, 210, 211

radiolabeling, 199, 211, 212

methylation interference assay, see

methylation protection/

interference, dimethyl sulfate

principle, 78

sites of reaction with DNA, 79

DMS footprinting, see Dimethyl sulfate

footprinting

DNA bending,

functions, 403

pBend vectors for assay,

bending angle, 415, 416

cloning sites, 405

colony screening for clones, 408, 411

electrophoretic analysis of

complexes, 408, 409, 412–414

host strains of bacteria, 409

materials, 405–409

overview, 403, 404

protein-binding site insertion into

plasmid, 405–411, 413

purification of plasmid DNA, 408,

411, 412

restriction sites, 404–406, 414, 415

types of vectors, 404, 405

DNase I footprinting,

applications, 31, 32

autoradiography, 36, 38

binding reaction, 35–37

combination with electrophoretic

mobility shift assay, 78, 79

digestion, 36, 37

DNA probe labeling, 34

DNase I,

sequence specificity, 33, 34, 79

structure and function, 32, 33, 79

gel electrophoresis, 36, 37

ligation-mediated polymerase chain

reaction for in vivo

footprinting,

advantages and limitations, 186, 187

cleavage for DNA sequencing

products,

A reaction, 201

624 Index

C reaction, 201, 202

G reaction, 201

overview, 200, 201

processing of samples, 202

reagants, 192

T+C reaction, 201

DNA polymerase selection, 191

DNA purification,

DNA extraction, 200, 213

materials, 191, 192

nuclei isolation, 200

quantification, 200, 213

DNase I treatment, 193, 204, 205,

213, 214

gel electrophoresis and

electroblotting,

blotting, 209, 214

electrophoresis, 208, 209

materials, 196, 197

hybridization,

digoxigenin-labeled probe,

197, 198, 210, 214, 215

materials, 197

radiolabeled probe, 197,

209, 214

ligation,

ligation reaction, 207

materials, 195

modified bases and conversion to

single-strand breaks, 188

nonspecific priming of 3'-ends,

188, 189

overview, 182, 186, 188

permeabilization of cells, 188

polymerase chain reaction,

cycles, 207, 208

materials, 195, 196, 213

primer extension,

incubation conditions, 206,

207

materials, 194, 195, 212, 213

single-stranded hybridization

probe preparation,

amplification product

purification and

quantification, 198, 199,

211, 215

digoxigenin labeling, 199,

212, 215

length, 210, 215

materials, 198, 199, 213

polymerase chain reaction

amplification, 198, 210, 211

radiolabeling, 199, 211, 212

materials, 35–37

nonspecific competitor DNA, 34

principle, 31, 77, 78

Southwestern blotting, see

Southwestern blot

titration, 34

DSC, see Differential scanning

calorimetry

Electron microscopy, see also Scanning

transmission electron microscopy,

nucleoprotein structural repeat

imaging,

focusing effects, 584, 585

materials, 571–573, 586

negative staining, 579, 580,

583, 584

overview, 579

specimen mounting, 583, 586, 587

tilted specimens,

applications, 580, 581

closer end identification,

584–586

Fresnel patterns, 585, 586

helical asymmetry

identification, 584–586

two-dimensional crystallography,