BioReg WorkshopAssaying Protein:DNA

Interactions

Immobilization/Migration Assays• Filter binding• Southwestern• MacKay• DNA affinity• Gel Shifts• Gel Filtration• Sucrose Gradient

Technique Overview

Protection Assays(positional information)

• Protection• Exo Protection• Modif-Interference• Genomic Footprint (in vivo)• Indirect Endlabeling (in vivo)

Electron Microscopy

• DS vs SS DNA; DNA unwinding• DNA wrapping around proteins• Protein conformational changes

Can Visualize

Crosslinking• Affinity enhancement for EM or

immobilization/ migration assays• Identifying specific contacts on protein

or DNA

Atomic Level Analysis• Crystal Structure• NMR

In Vivo Hints• One-hybrid• Allele specific 2nd site suppression

Things to Consider When UsingThese Techniques

• What is being followed: the protein or DNA?

• Which require pure proteins; which can be done with crude extracts?

• Which require significant occupancy of the binding site?

• How fast is the probing of the complex relative to dissociation rates?

• In those involving crude, which are convenient for purifying a protein?

• How is the DNA prepared for each assay?

• How much protein is needed for full occupancy?

• How do you show specificity? How do you determine what is recognized?

• Roughly how tight do the binding interactions have to be (off rates)?

• What are the limitations of each approach?

• How do you measure binding affinity (what are rough Kds for tight, mod, and weak binding)?

• How do you measure association rate or dissociation rate?

• How do you detect and measure cooperativity (2 dif proteins/1 protein)?

• How do you determine which part of a protein or which protein of a complex contacts the DNA?

• Which of these techniques is useful for showing protein induced DNA bending?

• Which of these techniques is useful for showing protein induced DNA looping?

• Which of these techniques is useful for showing protein induced DNA unwinding?

Filter Binding(DNA/pure/partial/rapid)

• Pure protein immobilization by adsorption to nitrocellulose filter

• Labeled DNA passes through unless bound to protein

• Useful for quantitating binding affinities and kinetics

• Note: all proteins retain binding ability when adsorbed to filter

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*

*

*

*

free DNA* *

**

proteinsprotein-DNA complexes

DNA Affinity Chromatography(protein/crude or pure/partial/slow-but very high on rate)

• DNA immobilized at high density to matrix by adsorption or covalent linkage• Bound proteins elute at higher salts• At lower salts high DNA conc favors rapid reassociation whenever protein dissociates• Useful purification step (proteins monitored by activity or western if Ab available)• Specific binding proteins separated from nonspecific binding proteins either by loading

column in competition with free nonspecific DNA or on the basis of higher salt elution required for specific binding proteins

• Variation: bind proteins to biotin labeled DNA before immobilizing DNA to streptavidin matrix lose advantage of large excess of DNA

low salt

load

high salt

elution

McKay (DNA/crude/partial/slow)

• Immobilize protein by immunoprecipitation• Labeled DNA bound to protein coIPs• Specificity determined by preferential coIP of specific DNA out of mixture containing

nonspecific DNA• Variations: cross-link before IP to counter problem of dissociation during washes detect

DNA by PCR

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*

* *

*

*

*

*

*

*

fragment with binding

site

inputDNA

IP’dDNA

Southwestern(DNA/crude or pure/partial/slow)

• DNA binding domain of protein renatures enough on nitrocellulose filter (after SDS-PAGE and Western) to allow for specific recognition of labeled DNA

• Useful for identifying which protein in a crude extract or in a tightly bound complex is responsible for DNA binding (proteins that do not bind offer additional specificity control)

• Assumption: a single polypeptide is sufficient for binding activity

probe with labeled

fragment*

autorad.

*

Gel Retention/Retardation/Mobility Shift (DNA/crude or pure/partial/“fast”)

• Protein binding retards motility of labeled DNA• Once complex has entered gel “caging” effect favors rapid reassociation

whenever protein and DNA dissociate (effectively reducing off rate)• Useful for following DNA binding activity during purification• Has also been used to quantitate binding affinities and kinetics• Multiple complexes can sometimes be seen as different shifted species• Note: not all complexes enjoy caging effect or are fully stable during time it

takes to enter gel

shifted DNA

free DNA

*

*

extract amount

Simple Shift ex: Multiple Shifts

Gel Retardation with Antibody Supershift:Use to identify proteins in gel-shift complex

shifted DNA

free DNA

* supershifted DNA

-Ab

+Ab

Gel Retardation with Protein Induced DNA Bending:If bending is induced, circularly permuted fragments will gel shift differently.

The closer the binding site is to the fragment center the greater the shift.

shifted DNA

free DNA

Gel Filtration (protein/pure/partial/slow)

• Large DNA molecules elute in void volume with any bound proteins• Free proteins elute in included volume

Free proteinINCLUDED VOLUME

DNADNA-protein

VOID VOLUME

Protein

DNA +

Bound Protein Free Protein

Fraction

Included VolumeVo

Sucrose Gradient(DNA/crude or partial/partial/slow)

• DNA coated with proteins sediments faster than naked DNA

• Useful for very large protein DNA complexes or extensive coating of DNA (e.g. chromatin association)

Sucrose SedimentationGradient

incr

easi

ng d

ensi

typrotein-coated

DNAnaked DNA

DNA

Increasing Size

Fraction dripped

from tube

bottom

Protection Assays• Gives positional information about the accessibility of a sequence to DNA

modifying/nicking/cutting probes• Position is defined with respect to a fixed reference site defined by a restriction cut or a

primer and so that:Fragment LENGTH = Modification/Nick/Cut POSITIONFragment length assessed at single nucleotide resolution by running on sequencing gel

For this length/position correlation to occur and to be informative:1) The DNA template must be labeled at only one end and on only one strand.2) modification/nicks/cuts must occur at most once per DNA molecule3) Inherent susceptibility of the DNA to the probe should be relatively independent of

sequence (and hence of position)4) Protein binding should cause significant decrease in probe susceptiblity of DNA

Uses: determine where a protein contacts DNA measure binding affinities and kinetics monitor multiple sites on a DNA molecule and quantitating cooperativity

Note: full to near full occupancy needed to see protection (exception: modif interference) may not see protection if protein-DNA interaction is dynamic

Standard Protection(DNA/crude or pure/full/fast)

Probes: DNase I

Dimethyl Sulfate (G,A modified, convert to nick with piperidine)

Copper Phenanthroline (attack on sugar)

Iron-EDTA (hydroxy radical attack on sugar)

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X

X

X

A B SEQ Gel

X

Interference(DNA/usually pure/partial/fast)

Modifying Probes: Dimethyl Sulfate (G, A); DEPC (A); KMnO4 (T); Ethylnitrosourea (phosphate); Formic Acid (depurination); Hydrazine (depyrimidation)

Only DNA in Protein-DNA complex is examined (isolated by preparative gel shift)DNA molecules modified at positions that interfere with protein binding will be excluded Modification is converted to nick after complexes are isolated

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Interference

bound DNA

free DNA

Genomic Footprinting(DNA/“in vivo”/full/fast)

Probes: Dimethyl Sulfate (permeable through live cells)DNAseI (have to lyse cells)

Analysis by:1) Primer extension with 5’ end-labeled primer; nick causes runoff at that position

(linear amplification from multiple rounds in thermocycler)2) PCR nicked fragments; details more complicated and not discussed (exponential

amplification from multiple rounds in thermocycler)

DNaseI nick primer extension to nick

protection

DNA-Protein Crosslinking-Identifying Protein Contacts by Label Transfer

(protein/pure/partial/fast)

• Crosslinking nucleotide is incorporated in DNA very close to a radiolabeled nucleotide• Crosslinking initiated by UV photoactivation (usually inefficient)• DNA is exhaustively digested with DNaseI leaving only crosslinked and neighboring nucleotides associated with

protein (and thereby labeling it)• Useful for identifying which protein in a complex or which segment of a protein (after proteolytic cleavage) is

near a specific nucleotide positionCrosslinking probe: T (has inherent ability to crosslink proteins when exposed to UV)

BrdU (significantly enhanced crosslinking over T)Photoreactive crosslinking arm attached to thiophosphate nucleotide

UV

Br*

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*

DNaseI

DNA-Protein Crosslinking -Identifying Protein Contacts by Peptide Transfer

(DNA/pure/partial/fast)

• Protein becomes UV crosslinked to nucleotides (particularly Ts) in DNA

• Protease digestion leaves nucleotide covalently linked to peptide

• Primer extension is blocked at modified nucleotide

UV

or

How would you demonstrate the following conclusions:

(in addition to ways discussed in the lecture, could you apply other techniques to show the same point?)

1) Ku binds blunt DNA ends

2) SSBP is cooperative

3) Beta subunit of Pol III is tightly bound to circular but not linear DNA

4) Gamma complex of Pol III can “load” beta onto a primer template or nicked DNA

5) Gamma complex of Pol III can also “unload” beta from primer template or nicked DNA

6) Core Pol III can prevent gamma complex from unloading beta from a primer-template (not from nicked DNA) but cannot prevent gamma from loading beta in the first place

7) RFC (clamp loader) binds to a structure (primer template junction) not a specific sequence

8) Largest subunit of the three protein RPA complex is responsible for the single-stranded DNA binding activity

9) oriC DNA wraps around approximately 30 dnaA proteins bound to the 9-mer elements

10) Nature of proteins assembled at yeast origins change during cell cycle (how would you try to identify the proteins responsible for the change?)

11) Which subunits of ORC contact the origin consensus sequence

12) CDC45 and the MCM proteins leave the origin and move down the DNA with the fork when elongation begins

13) Both early and late origins assemble pre-RCs during the M and G1 transition (can you say these pre-RCs are truly identical?)

• SV40 DNA replicated in vitro in the presence of CAF-1 and H3-H4 tetramer is preferentially coated with tetramers (as compared to unreplicated DNA)

• Sir proteins coat the ends of chromosomes from the telomeres to several kb in toward the centromere

• mutS recognizes and binds to mismatches (how would you determine if there is any mismatch preference?)

• mutS, mutL, and mutH may form a DNA loop

• E. coli RNAP binds -45 to +20 (what does it actually recognize?)

• Sigma factor is actually responsible for E. coli RNAP holoenzyme recognizing and binding -35 and -10 boxes

• E. coli RNAP undergoes a conformational change when it shifts from open to closed complex

• Sigma factor is not released from core RNAP and the DNA template until roughly 10 nt are polymerized (and core “clears” the promoter)

• lac repressor operators have different inherent binding affinities; lac repressor binds cooperatively to these sites

• CAP adjusts position of MalT binding to promoter of MalT regulon

• RNAP changes its interaction with the DNA template as it marches down the DNA

• LEF-1 (or HU, or TBP) induces DNA bending when it binds its site (how would you determine the angle of the bend?)

A Note on Measuring Binding Equilibria:

Kdis = [P][D]/[P:D]

where [P] is free protein conc, [D] is free DNA conc, and [P:D] is conc of protein-DNA complex

To measure Kdis you need to determine values for [P], [D], and [P:D]You control and hence know [Pt] and [Dt] in the experimentYou also know the following relationships: [Pt] = [P] + [P:D]

[Dt] = [D] + [P:D]

Most binding assays allow measure of [D]/[P:D] ratioe.g., % protection on footprint; % shifted up; % stuck to filter

Knowing the value of [Dt] you can use [Dt] = [D] + [P:D] and the measured ratio [D]/[P:D] to figure out [D] and [P:D]

Now that you know [P:D] you can calculate [P] from [P] = [Pt] - [P:D]

However . . .

In practice, Kdis can only be accurately determined if [Dt] is not greater than Kdis

Otherwise, if [Dt] is greater than Kdis, then the vast majority of any protein added will bind to the DNA (until the amount of protein reaches the amount of DNA). This can be inferred from the math. When the DNA is half occupied, [D] will be close in magnitude to [Dt] and thus greater than Kdis. In order for [D] x [P]/[P:D] to equal Kdis, [P]/[P:D] must be less than 1. Thus, [Pt] will be roughly equal to [P:D], and [P]= [Pt] - [P:D] will be the small difference of two much larger numbers and will not be accurately determined.

The best situation is if [Dt] is significantly smaller than Kdis so that [P]/[P-D] is large and [P] is approximately equal to [Pt]

You might think, then, that all you need to do is make sure [Dt] is much smaller than most Kdis you will be measuring (say 1pM).

However, for each assay there is a practical limitation of how low [Dt] can get based on how hot the DNA can be labeled and how much total counts are needed in the assay. For footprinting, [Dt] is often on the order of 0.1 to 1nM.

Corollary: Occupancy is not automatically determined by stoichiometry

Not all protein added to reaction will be bound to DNA

Percent association is determined by both DNA concentration and the value of Kdis

Only when [DNAt]>>Kdis, will occupancy be determined by stoichiometry, i.e. protein binding versus total protein concentration will be roughly linear.

Under these circumstances, in order for the product of [P]/[P-D] x [D] to be equal Kdis, [P]/[P-D] must be a small number, i.e. most of the protein that is added to the reaction will be bound to the DNA (the high DNA concentration is pushing the equilibria toward association)