Modeling DNA unzipping in the presence of DNA binding proteins

Farhat Habib, Dr. Ralf Bundschuh

Department of Physics,

The Ohio State University

Overview

Importance of understanding DNA-protein interactions

Single molecule experimental techniques The problem statement Theory and description of the applied model Results and conclusions Future directions

DNA

Carries genetic information Double stranded polymer

consisting of monomer units called nucleotides

4 nucleotides labeled A, G, C, and T

Basepairing A≡T, G≡C Each strand carries

complete information of the other

Proteins

Most diverse of macromolecules

Intermediaries in most biological reactions

Composed of smaller units called amino acids

Proteins play a vital role in DNA replication, transcription, recombination, repair, and in activating/inhibiting gene expression

Unzipping force analysis of protein association (UFAPA)

Single molecule experiment

Right sensitivity for probing DNA-protein interactions

F ~ 10 – 20 pN Distances ~ nm

Goals

To investigate the limitations of UFAPA The minimum binding energy for which the protein

can be detected Minimum distance between two proteins for which

they can be resolved

Theory and methods We describe the protein-DNA system’s

thermodynamic behavior or properties in terms of the partition function of the system

Model Break the DNA-protein system into two parts

The double stranded (ds)DNA with (or without) proteins

The single stranded (ss)DNA on which force is being applied

Model Partition function for dsDNA

The ssDNA In the highly stretched regime we will operate the

Extended Freely Jointed Chain (EFJC) model is the most accurate one

m

i

iE

N em)(

)(

Where E(i) is the stacking energy of the ith basepair

hRlml ehqR

hCmW pb /2)]([2

);(

R

Model (cont.) The protein

Include protein-DNA interaction by adding the extra free energy due to the presence of the protein at the binding site

Partition function for the entire system

where m0 is the protein binding site To obtain force at a given extension once we

have the partition function, we use

))(1)(;()()( /0

TkE

mNN

BprotemmmWmRZ R

)(log)( RZR

TkRf NB

Minimum protein strength

Top plot shows the force-extension curves from a protein of progressively lower binding energy at same position

Average force = 15.3 pN; Std deviation = 0.7 pN

At less than 10 kJ/mol the peak from the protein is within one standard deviation of the mean

Protein binding energy (kJ/mol)

GC:AT

20 40 60 80

1

2

3

4

25:75

50:5075:25

Cha

nge

in f

orce

(pN

)

30 kJ/mol

1300 1400 1500 1600 1700 1800

15

16

17

R(nm)

Fo

rce

(p

N)

-5 kJ/mol

30 kJ/mol

1300 1400 1500 1600 1700 1800

15

16

17

R(nm)

Fo

rce

(p

N)

-5 kJ/mol

Minimum resolvable distance between two proteins

14001500

1700

1800

50

40

20

10

121416182022

30

1600R(nm)

Prote

in s

epar

atio

n (b

p)

For

ce (

pN)

Averaged minimum resolvable distance

Minimum resolvable distance for pairs of proteins with 3 different relative binding energies

25 50 75 100

10

20

30

40

×2

×1

×½

Binding energy (kJ/mol)

Res

olva

ble

dist

ance

(ba

sepa

irs)

Conclusions and Future Directions We investigate the limits of the UFAPA technique by

considering the protein-DNA system thermodynamically Average force for bare DNA was found to be 15.3 pN with a

standard deviation of 0.7 pN Minimum binding energy for a protein to be detected using

UFAPA in the absence of FEC of bare DNA is around 10 kJ/mol

Minimum resolvable distance between two proteins can be up to 50 basepairs depending on relative protein strengths and the underlying DNA sequence

Future:Consider the kinetics of the process