Closing the Loop on Protein- DNA Interactions · Closing the Loop on Protein-DNA Interactions...

Closing the Loop on ProteinClosing the Loop on Protein--DNA InteractionsDNA Interactions

Institute for Mathematics and its ApplicationsUniversity of Minnesota

September 17, 2007

Stephen Levene, Ph.D.Departments of Molecular and Cell Biology and Physics

University of Texas at Dallas

AcknowledgmentsAcknowledgments

SupportSupport:: NIH (GM 47898, GM 55871, GM 67242), NIH (GM 47898, GM 55871, GM 67242), Texas Advanced Technology ProgramTexas Advanced Technology Program

SiteSite--specific specific RecombinationRecombination

Alexandre VetcherAlexandre VetcherAbbye McEwenAbbye McEwen

Farah BardaiFarah BardaiIsabel DarcyIsabel Darcy11

Yuri LyubchenkoYuri Lyubchenko22

Alex LushnikovAlex Lushnikov22

Yongli ZhangYongli Zhang88

Don CrothersDon Crothers66

DNA Topology and SupercoilingDNA Topology and Supercoiling

Hua TsenHua TsenIsabel DarcyIsabel Darcy11

Andreas HankeAndreas Hanke77

Rob SchareinRob Scharein55

RepeatedRepeated--sequencesequenceDNADNA

Sima ZeinSima ZeinRichard SindenRichard Sinden33

Woody WrightWoody Wright44Jerry ShayJerry Shay44

1Dept. of Mathematics, Univ. of Iowa 7Dept. of Physics and2Dept. of Pharm. Sciences, Univ. of Neb. Med. School Astronomy, UT-Brownsville3IBT, Texas A&M Univ. 8Phys. Biosci. Div., Lawrence4UT-Southwestern School of Med. Berkeley Laboratory5Hypnagogic Software, Vancouver, BC6Depts. of Chemistry, Mol. Biophys. & Biochem., Yale Univ.

15-5

0 kb

picoscalenanoscale mesoscale

DNA Biophysics in the 21DNA Biophysics in the 21stst

CenturyCentury

• Role of DNA looping in biology

• General principles of DNA looping deduced from statistical mechanics

• Applications to loop-mediated gene repression: the lacoperon

• Looping in site-specific recombination and implications for DNA topology

OutlineOutline


DNA Looping in BiologyDNA Looping in Biology

• Gene regulation

Repression: ara, deo, gal, lacoperonsActivation: glnALG operonMany eukaryotic examples (e.g., β-globin locus)

• Intramolecular site-specific recombination (Int, Cre, Flp, Gin/Hin, etc.)

• Transposition (Mu)

• Type-II restriction enzymes (SfiI, NgoMIV)

• DNA mismatch repair (MutHSL)


Courtesy of Mike White, UCSD

DNA size, bp

103

J, m

ol L

-1

10-9

10-8

10-7

200

A

DNA size, bp235 240 245 250 255

J, m

ol L

-1

10-9

10-8

B

Relationship to DNA CyclizationRelationship to DNA Cyclization

Levene, Nature ELS (http://www.els.net)


• J drops precipitously with DNA size for small DNAs

• Near 200 bp, J varies by ~100-fold over a full helical turn

• J values are extremely sensitive to the presence of intrinsic bends

Solving the DNASolving the DNA--looping Problemlooping Problem


( )21 3

21 1

Nij ij

i j ij

x xHβ

σ

−

= =

−= ∑∑Hamiltonian:

= instantaneous rotation angle (tilt, roll, or twist) of the i-th rigid body relative to (i-1)-st

= variance of rotation angle

= corresponding mechanical-equilibrium angle

ijx2ijσ

ijx

{ }( )( ) : 1, , 1; 1, ,3 0; 1, ,6kijf x i N j k= − = = =… … …Non-linear constraints:

-2

6B

8 e expkdet( )det( )

sEloopG

JT

π

π

Δ⎡ ⎤= = −⎢ ⎥

⎣ ⎦A F

Harmonic-approximation solution for the J factor:

• The J factor is:

Proportional to Keq for the formation of a closed loop from an open chainEffective concentration of one loop end in the vicinity of anotherRatio of statistical-mechanical partition functions for closed and open loops

Zhang & Crothers Biophys J

(2003)


Parameterization of DNA Parameterization of DNA ConformationsConformations

Tilt

θ

Roll

φ

Twist

τ


φDPτPP ≈ 0

φPD

τPP = -60°

τPP = -60°

Zhang et al. Biophys J (2006)


1 6 8 1 7 2 1 7 6 1 8 0 1 8 4 1 8 8 1 9 2

1

1 0

1 0 0

(b(a )N

J

fact

or (n

M)

D N A leng th (bp )

0 ° 6 0 ° 1 2 0 °

1 6 8 1 7 2 1 7 6 1 8 0 1 8 4 1 8 8 1 9 2

1

1 0

1 0 0

(b(a )N

J

fact

or (n

M)

D N A leng th (bp )

0 ° 6 0 ° 1 2 0 °

Coupling of Twist and Writhe in Coupling of Twist and Writhe in DNA LoopingDNA Looping



Effects of DNAEffects of DNA--sequence Symmetrysequence Symmetry

1

10

100

168 172 176 180 184 188 1920.1

1

10

100

J fa

ctor

(nM

)

+90 -90 Sum

(d(c)

(b(a)

J fa

ctor

(nM

)

DNA length (bp)

+45 -135 Sum


• Two distinct looped conformations contribute to the J factor• Dramatically reduced phase dependence – cannot rule out looping

Summary Summary –– Part IPart I

• Numerical approach to computing looping free energies based on harmonic approximation is 104-fold more efficient than Monte Carlo simulation

• DNA looping is distinguished from cyclization by strong coupling of twist and writhe

• Relationship between Tw and Wr in small loops can generate phase shifts such that the most energetically favorable loops involve non-integral numbers of helical turns

• Negligible helical-phase dependencies do not necessarily imply absence of DNA looping



Intact E. coli cellV ≈ 1 fL = 1·10-12 cm3

50x to 100x

≈1 μ

m

E. coli genome4.6·106 bpL = 1.6 mm

= 6·10-11 cm3

2 3 / 24 3V S= < >

Architectural DNAArchitectural DNA--bending Proteins in bending Proteins in Genome Organization and RegulationGenome Organization and Regulation

HU-DNA cocrystal structureSwinger et al. Embo J (2003)

E. coli architectural DNA-bending proteins:

HU ≈ Fis > IHF > H-NS > StpA > Dps

O1

O3

O1

O2

O1O3 O2

92 bp 401 bp

4 (LacR)

DNA Looping and Regulation of DNA Looping and Regulation of the the laclac OperonOperon

• Looping between the primary operator, O1, and auxiliary operators, O2and O3, enhances repression by increasing the effective concentration of LacR at the promoter


+

Structure of the Structure of the LacRLacR TetramerTetramer


y

xH

C

T

DP/PD PD/DPPP

“V-shaped” tetramerLewis et al. Science (1996)

“Extended” tetramerRuben & Roos Microsc Res Tech (1997)

137-bp DNA loop mediated by extended tetramer

PDPP

DP

The The ““VV--shapedshaped”” Repressor Forms Repressor Forms Multiple Loop Geometries Multiple Loop Geometries


J = 0.42 nMΔGloop = 53.5 kJ mol-1

“WT”179 bp

(+), J = 0.01 nMΔGloop = 62.8 kJ mol-1

(-), J = 2.1 nMΔGloop = 49.5 kJ mol-1

“WA”179 bp

“WA”163 bp

(-), J = 0.75 nMΔGloop = 52.0 kJ mol-1

(+), J = 0.78 nMΔGloop = 52.0 kJ mol-1

“LB”163 bp

(-), J = 15 nMΔGloop = 44.6 kJ mol-1

0 nMJ ≅(+),ΔGloop > 100 kJ mol-1

Zhang et al. PLoS One (2006)

The Extended The Extended LacRLacR Tetramer is Tetramer is the Dominant Form in Small Loopsthe Dominant Form in Small Loops


DNA length (bp)60 80 100 120 140 160 180

J (n

M)

0.001

0.01

0.1

1

10

100

1000

SL (15,30) LB (0,0)Sum

-ΔG

0 loop

/kBT

-28

-26

-24

-22

-20

-18

-16

-14

DNA length (bp)130 140 150 160 170 180

J (n

M)

0.001

0.01

0.1

1

10

LBWAWTSum

-ΔG

loop

/kBT

-26

-24

-22

-20

-18


Thermodynamic Model for Thermodynamic Model for LacRLacRRepressionRepression


12

12 1212

12

K1 K2

K2 K1

Kc(1)Kc

(2)

1 2 1 2( )c t

t t t

d JPD K K K K J P P

λλ

=+ + + +

( )( )1 2

1 1noloop t

loop t t

E JPR JE K P K P

λ= = + ≡ + Γ

+ +

( )( )

1 2

1 2 1 2

tloop

t t

K K PE

K K K K J P Pλ+

=+ + + +

1

1noloop

t

KEK P

=+

1 2( )( )t

t t

PK P K P

λΓ =

+ +

0( ,DNA flex., protein flex.)J J h=

(known)

(4 params)


Analyzing Experimental DataAnalyzing Experimental Data


Data of Müller et al.J Mol Biol (1996)

Data of Becker et al.J Mol Biol (2005)


Analyzing Experimental Data Analyzing Experimental Data (cont(cont’’d)d)


16 (± 1)10.95 (± 0.03)0.8 (± 0.1)128 (± 2)1.04.66b25Becker et al. [18]ΔHU

19 (± 1)11.08 (± 0.04)0.7 (± 0.1)95 (± 3)1.14.66b26Becker et al. [18]WT

20.7 (± 0.5)11.60 (± 0.01)1.1 (± 0.1)95 (± 1)1.01.17a51Müller et al. [5]

Protein flexibility,

deg.

Helical repeat,

bp·turn-1

Torsional rigidity, 10-19

erg·cm

Persistence length, bp

Best-fit valuesFitting errorΓ, x 102

No. of data

points,Nd

Data Set


Looping, Looping, LacRLacR flexibility, and HUflexibility, and HU--dependent Bending Dramatically dependent Bending Dramatically Increase Repression EfficiencyIncrease Repression Efficiency

©© 2007 S.D. Levene. All rights reserved.2007 S.D. Levene. All rights reserved.Zhang et al. PLoS One (2006)

Summary Summary –– Part IIPart II


• Small loops ( 200 bp) between lac operators are predominantly mediated by the extended LacR tetramer conformation

• LacR-dependent regulation in vivo is facilitated by enhanced DNA flexibility in the presence of HU protein

• HU binding and protein flexibility are both important factors that promote DNA looping over short distances

<

3' 5'

Mechanism of Tyrosine SiteMechanism of Tyrosine Site--specific specific RecombinasesRecombinases

a b

a b

a b

a b

Cleavage, strandexchange

Isomerization (?),HJ resolution

X

a b

a b

5' 3'

5' 3'

5'3'

Holliday-junctionintermediate


3-noded knot3-noded knot

DNA Knotting via SiteDNA Knotting via Site--specific specific RecombinationRecombination


well

unknotted,nicked plasmid

7-noded knot5-noded knot

9-noded knotsc plasmid

linear plasmid4-kb linear

3-kb linear

kb-ladder

supercoiled plasmid

kb-ladder

knotted plasmid

supercoiled plasmid

knotted plasmid

linear p

lasmid

unresolvedknots

=

Electron micrograph of a (+3) DNA knot Tsen & Levene, unpublished

Topology of Topology of CreCre Recombination is Recombination is Inconsistent with a Planar IntermediateInconsistent with a Planar Intermediate


TATTGAAGCATATCGTA

ATAACTTCGTATAGCAT ACATTATACGAAGTTAT

TGTAATATGCTTCAATA

ATAACTTCGTATAATGT

TATTGAAGCATATTACA

ATGCTATACGAAGTTAT

TACGATATGCTTCAATA

(11)abθ

(12)baθ

TATTGAAGCATATCGTA


TGTAATATGCTTCAATA

ATAACTTCGTATAATGT

TATTGAAGCATATTACA

ATGCTATACGAAGTTAT

TACGATATGCTTCAATA

TATTGAAGCATATCGTA

ATAACTTCGTATAGCAT

TATTGAAGCATATCGTA


TGTAATATGCTTCAATA

ACATTATACGAAGTTAT

TGTAATATGCTTCAATA

ATAACTTCGTATAATGT

TATTGAAGCATATTACA

ATAACTTCGTATAATGT

TATTGAAGCATATTACA

ATGCTATACGAAGTTAT

TACGATATGCTTCAATA

ATGCTATACGAAGTTAT

TACGATATGCTTCAATA

(11)abθ

(12)baθ

Cre-HJ cocrystal structureGuo et al. Nature (1997)

AtomicAtomic--force Microscopy of force Microscopy of CreCre Synaptic Synaptic ComplexesComplexes


(80%)

(97%)

Vetcher et al. J Mol Biol (2006)

ConclusionsConclusions


• Development of comprehensive theory for DNA looping that accounts for DNA and protein conformation, protein flexibility, thermal fluctuations, and helical phasing

• Small regulatory loops ( 200 bp) in the lac operon are mediated by the extended LacR tetramer conformation and regulation in vivo is facilitated by enhanced DNA flexibility in the presence of HU

• Understanding DNA looping is vital for rigorously interpreting results of topological experiments

• Loop-closure kinetics is an emerging tool for analyzing the structure of complex nucleoprotein assemblies

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Nick Cozzarelli1938 - 2006

Closing the Loop on Protein- DNA Interactions · Closing the Loop on Protein-DNA Interactions...

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