Fluctuational Opening of Base Pairs in DNA

Maxim Frank-KamenetskiiBoston University

mfk@bu.edureprints at: www.bu.edu/cab

Two types of interactions stabilize the DNA double

helix: base pairing and base stacking

Being a nanoscale object, DNA must experience

thermal fluctuations, or breathing:

Example: 3 base pairings and 4 base-

stackings are disrupted

Why is it important?• Due to the breathing, reactive groups normally

buried inside the double helix become accessible for chemicals and proteins

• Nuclear magnetic resonance (NMR) makes it possible to study base-pair breathing for very short DNA helices thus providing with direct experimental data to compare with a prediction algorithm

• Base-pair opening is very important for DNA damage, mutations and repair

Example: Uracil DNA Glycosylase flips out uracil from the double helix

Parikh et al. PNAS, 2000

•Base-pair opening is very important for DNA damage, mutations and repair

Watson - Crick

Base Pairs

imino protons

To separate base-pairing and base-stacking contribution into the

melting free energy, one needs to determine independently either

stacking or base-pairing

BPL

BPK

STKLKL GGGG

2

1

2

1

•Base pairing: GBPA and GBP

G

•Stacking: GSTKL

DNA stability with respect to melting

Nicked DNA

X-ray crystallography, NMR, EM, PAGE, thermal denaturation studies agree:

Nick introduces only minor perturbations to the structure of the DNA

double helix.

Really?

DNA fragments with solitary nicks or gaps

30 0 bp fragm ent

10 4 bp

PvuI I PvuI I

Nicks are introduced enzymatically by nicking endonucleases

Nicks are located in the KL/K’L’ dinucleotide stack in the DNA fragment

30 0 bp fragm ent

~ 10 0 bp

PvuI I PvuI I

Gaps (2-nt or longer) are obtained by consecutive digestion by two nicking enzymes

N.BstNBI N.BbvCIA

N.BstNBI

N.AlwI

Gel electrophoresis of nicked DNA

2.3 M

3.5 M

4.9 M

7.0 M

GGCC

ACTG

TCAG

CGGC

AGTC

2 bpgap

10 bpgap

5 '

3 ' GCCG

FORREV M

DNA fragments carrying a single nick

• are somewhat retarded with respect to intact fragments• retardation is enhanced by addition of denaturant (urea) to the gel• degree of retardation dependson the identity of nicked dinucleotide stack

1 2 3 4 5 6 70.80

0.85

0.90

0.95

1.00 pGC

pCC

pCA

pTA

pG2

Urea concentration, M

Rela

tive m

ob

ilit

y

Stacked-Unstacked Equilibrium in nicked DNA

=

RT

G

N

N ST

open

closedexp

Stacked or closed conformation:straight DNA molecule; moves fast

Unstacked or open conformation:

kinked DNA

closed open

closed

open

=-

-

is the gel-electrophoretic mobilityre

l ati

ve m

ob

i lit

y

0.70

0.75

0.80

0.85

0.90

1.2 M

1 2 3 4 5 6 7

gap size, nt

We assume that the kinked molecule moves like gapped DNA:

GST

Standard free energies are estimated by extrapolating

GST(urea) to zero

0 2 4 6-2

-1

0

1

0 2 4 6 0 2 4 6

GC GG AC

0 2 4 6

TC

0 2 4 6

CG

0 2 4 6

AG

0 2 4 6

TG

0 2 4 6

AA

0 2 4 6-2

-1

0

1 AT

0 2 4 6

TA

0 2 4 6

GA

0 2 4 6

GT

0 2 4 6

CC

0 2 4 6

TT

0 2 4 6

CA

0 2 4 6

CT

Fre

e e

nerg

y

calc

ula

ted

fro

m

mob

ilit

y d

ata

Urea concentration, M

GKLGCKLC''

GKLGCKLC''

We obtain 32 stacking parameters

GC GG CC CG AC GT TC GA AG CT TG CA AT AA TT TA-3

-2

-1

0

G

KL

ST

, kca

l/mol

GST values describing the equilibrium at the site of the DNA nick are equivalent to the stacking parameters of intact double helix

-GKLG--CKLC-''

-GKLG--CKLC-''

Determination of base-pairing contribution to melting free

energy

BPL

BPK

STKLKL GGGG

2

1

2

1

•Base pairing: GBPA and GBP

G

•Stacking: GSTKL

0.0 0.2 0.4 0.6 0.8 1.0340

350

360

370

380

390

400

me

ltin

g t

em

pe

ratu

re,

K

GC content

Marmur-Doty plot

DNA differential melting curves

Free energy of DNA melting GKL

BPL

BPK

STKLKL GGGG

2

1

2

1

•Base pairing: GBPA and GBP

G

•Stacking: GSTKL

I1 = GAA

I2 = GGG

I3 = GAT + GTA

I4 = GGC + GCG

I5 = GAC + GCA

I6 = GAG +GGA

I7 = GAT + GTC + GCA

I8 = GAG + GGC + GCA

There are 16 KL contacts, 10 of them are different;

only their 8 invariants can be determined from melting experiments with long DNA molecules:

in

va

ria

nt

, k

ca

l/m

ol

I I I I I I I1 2 3 4 5 7 86

-2.5

-2.0

-1.5

-1.0

-0.5

invariants

I

Temperature Dependence

individual stacks

TAAT

TAAT

AATT AATT

22 °C

32 °C

42 °C

52 °C I

I

I

I

G

G

G

G

N

N

N

N

temperature, °C

TAATAATTATTA

10 20 30 40 50

-2.0

-1.5

-1.0

-0.5

0.0

0.5

G

, kca

l/mol

ST

temperature, °C

CGGCGGCC

GCCG

30 35 40 45 50 55

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

G

, kca

l/mol

ST

F ig u re 6

G

, kc

al/m

olBP

a

b

fre

e en

ergy

, kca

l/mol

temperature, °C

temperature, °C

GST

G

A T

G C

10 20 30 40 50-2.0

-1.5

-1.0

-0.5

0.0

10 20 30 40 50-1.0

-0.5

0.0

0.5

1.0

Temperature dependenceof stacking andbase-pairing

Salt dependenceof stacking andbase-pairing

F ig u re 7

G

, kca

l/mo

lBP

a

b

free

ene

rgy,

kca

l/mol

sodium, mM

sodium, mM

G C

A T

10 100-0.5

0.0

0.5

1.0

1.5

10 100

-2.0

-1.5

-1.0

-0.5

0.0

Theoretical prediction of bp opening probability

RT

G BPi

i exp

1,0 1,0 1

),(),(1

1,0 2

11

1

)()()()(......

N

iiiiii

N

i

ffiii

1,0 1,0 1

),(),(1

1,0 1,01,0 2

11

1 1

)()()()(.........

N

iiiiii

k ki

N

i

ffiii

= Kd

RT

G STii

i,1exp

k

k k+1

Base-pairing

Stacking

Ring factor (adjustable parameter)

Z

1,0 1,0 1

),(),(1

1,0 2

11

1

)()()()(......

N

iiiiii

N

i

ffiiiZ =

Partition function for

DNA:

DNA BreathingNMR

Theory

NMR data from

Gueron & Leroy and

Russu groups

C C T3 T4 T5 C60

10

20

30

40

i

G G A3 A4 A5 G60

10

20

30

40

iiC G C3 A4 C5 A6

0

10

20

30

iv

C G C3 A4 G5 A60

10

20

30

iii

bp o

pen

ing

prob

abili

ty x

10

6

bp o

peni

ng p

roba

bili

ty x

10

6

G C G3 A T5 C6 T G8 T9 T10 C11 T12 A13 T T15 G C0

10

20

30

40

vibp

ope

ning

pro

bab

ility

x 1

06

G C G3 A4 T C6 T A8 T9 T10 T11 A12 T13 T14 T15 G C0

5

10

15

20

v

Temperature dependence of bp opening

C3 A4 G5 A6 C3 A4 G5 A6 C3 A4 G5 A6 C3 A4 G5 A6

0.5

1.0

1

2

3

4

5

10

15

0

10

20

30 bp

op

en

ing

pr

ob

ab

ility x

10

5

5 °C 15 °C 30 °C 37 °C

= 2.3·10-3

for all temperature

s

NMR (Russu group)

Theory

• Conclusions• Stacking rather than base-pairing determines

the stability of the double helix with respect to melting

• DNA breathing probability can be predicted by an algorithm based on the partition function calculation

• Since stacking dominates, the opening probability strongly depends on the nearest neighbors

Acknowledgements

• Katya Protozanova

• Peter Yakovchuk

• Andy Krueger