A novel loop structure observed in d-GAATTCCCGAATTC by 2D NMR

Vol. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 30, 1986

Pages ]224-1232

A NOVEL LOOP STRUCTURE OBSERVED IN d-GAATTCCCGAATTC BY 2D NMR R.V. Hosur, Anu Sheth, K.V.R. Chary, M. Ravikumar~and Girjesh Govil Chemical Physics Group, Tata Institute of Fundamental Research Homi Bhabha Road, Bombay 400005, India and Tan Zu-kun and H. Todd Miles National Institutes of Health, Bethesda, Maryland 20205 Received August 5, 1986

SUMMARY: Sequential resonance assignments of the non exchangeable base and sugar protons in d-GAATTCCCGAATTC have been obtained using two dimensional NMR experiments at 500 MHz. The chemical shifts and the NOEs have been used to determine the structure in the base-pair mismatch region which is located in the central portion of the molecule. It is observed that the molecule adopts a novel unsymmetrical loop structure in this section which is characterised by sugar geometries which are significantly different compared to the rest of the molecule. The base-paired portion of the molecule conforms to a right handed B-DNA type of structure. © 1986 AcademicPress, Inc.

The double helical right handed DNA models A and B, have played a major interpretive role in molecular biology during the last three decades. In recent years, it has become increasingly evident that DNA can show significant deviations from these forms depending on factors such as molecular weight, base composition and sequence, experimental conditions, etc. Dimensional

(1-9).

Two

(2D) NMR spectroscopy which has been used to determine solution

structures of DNA segments, 10-15 units long, has contributed significantly in this regard.

Such deviations in the secondary structure of DNA can act as

recognition and functional sites in the molecule.

*To whom correspondence should be addressed. Abbreviations Used: NMR, Nuclear Magnetic Resonance; NOE, Nuclear Overhauser Enhancement; DNA, Deoxyribo Nucleic Acid; COSY, Correlated Spectroscopy; COSS, Correlation with Shift Scaling; NOESY, NOE Correlated Spectroscopy; TSP, Sodium 3-trimethyl Silyl (2,2,3,3-2H) propionate; ppm, Parts Per Million.

0006-291X/86 $1.50 Copyright © 1986 by Academic Press, lne. A II rights of reproduction in any jorm reserved.

1224

Vol. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In the p r e s e n t paper, ral studies

assignments

and structu-

on

1 5' - d-G

2 A

c a r r i e d out using molecule,

we report the resonance

4 T

2D COSY

hereafter

form base pairs.

3 A

6 C

7 C

(i0), COSS

labelled

This

5 T

8 C

9 G

I0 A

(ii) and NOESY

as 14-met,

12 T

13 T

molecule.

We show here that the m i s m a t c h

variations

in the molecule.

14 C - 3',

(12) experiments.

has two extra cytosines

leads to a m i s m a t c h

MATERIALS

ii A

in the central

which cannot

portion

leads to important

This

of the

conformational

AND M E T H O D S

The 14-met has been s y n t h e s i s e d using standard p r o c e d u r e s (13). NMR spectra have been r e c o r d e d using a 4 mM s o l u t i o n in D~O (pH 7.2) at 25°C on a Bruker AM-500 NMR spectrometer. Phosphate buffer has been used to m a i n t a i n pH, with the total Na ion c o n c e n t r a t i o n being 0.1M. COSY and NOESY data have been collected with 512 t I and 2048 t 2 points and the time domain data was F o u r i e r t r a n s f o r m e d after w i n d o w m u l t i p l i c a t i o n by sine square bell and sine bell functions, along t and t I axes respectively. The COSS experiments have been c a r r i e d out with d ~ f f e r e n t shift scaling factors for a p p r o p r i a t e r e s o l u t i o n e n h a n c e m e n t and a c c o r d i n g l y the number of tl experiments was optimised. C h e m i c a l shifts have been e x p r e s s e d in ppm with respect to TSP. RESULTS Assignments gonucleotide 5, 9, 14,

have been o b t a i n e d

shown

identified

resolution to HI'

in Fig.

particular

--i.

(H2', H2")

units.

2-4 enable

H2' and H2" protons, elsewhere

spectrum,

and H2'

of cytosines

of COSS

resonance

spectra

corresare

of the molecule.

1225

to

using the distance

unit as well as on the adja-

NOESY c o n n e c t i v i t y

assignment

manner.

and

cross peaks b e t w e e n base

diagrams

shown

of base and sugar HI',

The details

and in the f o l l o w i n g we use these a s s i g n m e n t s

about the structure

In the

--- H2" cross peaks

which contains

The i n t e r n u c l e o t i d e

in a s e q u e n t i a l

(4,

COSS spectra have

of the m o l e c u l e

on the same n u c l e o t i d e

unambiguous

strategies

above.

and base protons

of the oli-

the spin systems have been assigned

along the sequence

in the NOESY

cent n u c l e o t i d e

mentioned

two sections

cross peaks

In the second step,

and sugar protons

in Figs.

the well d o c u m e n t e d

from COSS and COSY spectra.

and as an i l l u s t r a t i o n

nucleotides

correlations protons

following

base and sugar protons

all the sugar ring spin systems

thymines were

ponding

of the non e x c h a n g e a b l e

15) based on the use of 2D e x p e r i m e n t s

first step,

better

AND D I S C U S S I O N

will be p u b l i s h e d

to derive

information

Vol. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5[

,

b

~ C8

1"8 C6

2"2

~

T4

~ C8

,~TI3

I

2"6

.,,~.

.,,o & , t " ,,O ~,,o 0o,

3"0

Ac, ',6o o"

o

I

I 64

I 60

,

I 5'6

3'0

2-5

1 (a).

(b) .

Section of the 500 MHz COSS spectrum of the 14-mer showing HI'(H2', H2") cross peaks. Each HI' proton gives rise to t w o c r o s s peaks indicating reasonably large values (6-7 Hz) of HI'-H2' and HI'--H2" coupling constants. 512 t I and 2048 t_ points were used for data collection. Shift scaling factor is i~5. Section of 500 MHz COSS spectrum showing H2'--H2" cross peaks. Shift scaling factor is 2. The experiment has been optimised to reduce the diagonal peaks so that cross peaks close to the diagonal can be observed. 256 t I and 2048 t 2 points were used.

,'I i#" III

'~i II

l,z

,K

IJ

',

''

J

Jl

I "I I

L 7,0

7-5

H8/H6 8"0

I

8.0

I

7-5

7-0

H8/H6 Fig. 2.

, ]-5

H2"

HI' Fig.

2-0

Section of the 500 MHz NOESY spectrum of the 14-mer showing sequential (H8/H6) .---(H8/H6. +4 connectivities. The spectrum also reveals unexpected connectlvl~les C8 < - - > C6 and G9 < - - > C6 (dashed lines) which reflect short base - base distances between these pairs of nucleotides and hence provide evidence for loop structure in the centre of the molecule. Mixing time = 500 msec.

1226

Vol. 1 3 9 , No. 3, 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL

II1

I I

,,

in

',]

I

~G9 I

'[

'I

RESEARCH C O M M U N I C A T I O N S

I

I

o .A

c6

• 6"0

A5 ,,

t

T13

8-0

7.5

H8/H6 Fig. 3.

Section of the 500 MHz NOESY spectrum (Mixing time = 400 msec.) showing (H8/H6) --- (HI') . . connectivities. The assignment of the 1 . lbase protons is given on t~e top of the figure while that of the HI' protons is given on the horizontal lines in the connectivity path. There are also two interstrand connectivities (.dashed lines) namely A3 H2 TI2 HI' and All H2 - - T4 HI' Besides, a connectivity between H5 of C14 and H6 of TI3 is also seen in the figure~

The NOESY cross peaks enable us to place a number of intra and interstrand d i s t a n c e structure

constraints

of the 14 mer.

and p r o v i d e

For example,

a fairly good idea about the s e c o n d a r y the cross peak b e t w e e n H2 p r o t o n of A3

and HI' p r o t o n of TI2 and the cross peak b e t w e e n H2 of All and HI' of T4 (Fig.

3) indicate

that A3 and All n u c l e o t i d e s

on one strand are base p a i r e d

to TI2 and T4 residues on the complementary strand, internucleotide

connectivities

--(H2', H2")i_l

in Fig.

indicate

(H8/H6) i- (H8/H6)i±I,

2-4 and the c o n n e c t i v i t i e s

that the -AATT- segments

the other hand, seen,

namely,

These

indicate

<

(HI')i_I,(H8/H~i

from methyl p r o t o n s

long range NOE c o r r e l a t i o n s

(Fi~4) On are

> C8(H6),C6(H6) <-->G9(HS) andC7(H5)<-->T5(CH3).

a major d e v i a t i o n

from the regular helical

central part of the m o l e c u l e w h i c h contains also i n d i c a t i v e

(H8/H6)i-

Further, the

adopt an o v e r a l l B-type of structure.

in the i n t e r v e n i n g portion,

C6(H6)

respectively.

of loop s t r u c t u r e s

the b a s e - p a i r

in the m i s m a t c h

1227

structure mismatch.

in the They are

region of the molecule.

Vol. 139, No. 3, 1986

<<<<

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

',i

oo

,i

C ,

]1

I f

I

".,[#'" tl H

~ C E , , I

I

ql'O

i

a

A11 A3

T T , ~ : ~

15

CH3

T5 (CH3) ,=,-- - ~

,i

~C 8

C7 (HS) o C6

k)°

~ T4

rsO00'r,2

C8

o

2.0 o~

I

C7

.

I , ~ ~

~

,

T 5 , ~ L ~ C7

G1

25

,¢rt

~IW 30

t

I

i

7.5

8.0

I

7.0

I

6-5

H 8/H6 Fig. 4.

Based I,

on these

II a n d

III

bonds

per

backbone from

type

considerations (Fig.

strain

5).

whereas of

dihedral

one

III

is t h e angles

point

has

same.

I and

the

II

these

three

involve

loop

1228

bends

out

the

region

from

angles of

of

loop

two molecules

structures,

different

C8

types

which

the bend

dihedral

I, C7 a n d

three

molecule

However,

significantly

In

visualise

a single

In a l l

of view,

in t h e m o l e c u l e .

can

Structures

structure.

molecule

energetic

5.5

Section of the NOESY spectrum showing H8/H6---(H2', H2") cross peaks, HI'---(H2',H2") cross peaks and H8/H6---CH. cross peaks J (box a on top left). Sequential connectivities via H8/H6/H5 - (H2", H2') protons have been drawn in the figure. Except in the Stretch C8-C7-C6-T5, the connectivities have been shown via H2" proton. In the above mentioned stretch H2' proton has been used. The H2', H2" proton chemical shifts used in the connectivity have been marked on the horizontal lines, while the assignment of base protons is given on the top of the figure. The HI'---(H2', H2") cross peaks have been identified by the respective nucleotide symbols. In every case the downfield peak corresponds to the H2" proton. The right half of the spectrum also shows a cross peak between C7 H5 and T5 CH 3 protons.

in b a s e - p a i r i n g hair-pin

I

6.0 H5/HI'

of 1 4 - m e t forms

number

in III w i l l

those

may

and

structures

in

produce

I and

a

o f Hhave II,

an extra

the helix resulting in a

and

Vol. 139, No. 3, 1 9 8 6

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

7

8

C--C 1

2

S 4

5 61

19 10 11 12 15 14

G- A-A-T-T-C

G-A-A-T-T-C

d-t-+-i i G d-t-t-i-i-~ 14 13 12 11 1091

16 5

4

3

2

1

C--C 8

7

6

7

C--C 1 2 3 4 51 18 9 I0 11 12 13 14 G-A-A-T-T C-G-A- A-T-T- C IT

;

:

,

i

,

:

,

,

I

C - T - T - A - A - G-C T-T-A-A-G 14 15 12 11 10

9 81

15 4

5

2

1

C-C 7

]

2

5

4

5

6

7

G - A - A - T - T - C- C '1 i.i . i,. . ii . 1 i I C-T-T- A-A-G-C

TIT

14 15 12 11 10 9 Fig. 5.

symmetrical result

double

stranded

The o b s e r v a t i o n

hydrogen-bonded above

structure.

In II, C6 and C7 units

structure.

allow d i s c r i m i n a t i o n

ghtaway eliminates

between

of h a i r - p i n

G-C base pair,

shifts

of the HI',

H2'

consistent

with the well known o b s e r v a t i o n

which

respect to HI'

Likewise

to HI' of All and HI' p r o t o n s of T5 and TI3 respectively.

observation

nucleotide

made earlier

upfield

since

spectrum.

that purine

is flanked by G1 and A3 appears

(Fig.

2) straiin the usual

the two p r o t o n s m e n t i o n e d

la that the HI' chemical

shifts on n e i g h b o u r i n g

is flanked by A2 and T4.

(III),

and base p r o t o n s p r o v i d e

It is seen in Fig.

of A2 which

between

in the NOESY

criteria.

large ring current

structure

the distance

experimen-

these structures.

of NOEs b e t w e e n H8 of G9 and H6 of C6

possibility

loop out and

In the f o l l o w i n g we p r e s e n t

is too large to give a cross peak

chemical

8

Possible types of loop structures in d-GAATTCCCGAATTC.

in an u n s y m m e t r i c a l

tal facts which

6

The relative

further

selection

shifts are g e n e r a l l y rings

units.

Thus HI' proton

compared

HI' of AI0 appears

(A and G) produce

to HI'

of A3

u p f i e l d with

of T4 and TI2 appear u p f i e l d compared

T h e s e patterns

are c o n s i s t e n t

that the two ends of the m o l e c u l e

1229

with the

form a regular

Vol. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

double helix.

the cytosines,

As regards

to behave d i f f e r e n t l y is o b s e r v e d

in Fig.

compared

to the base p a i r e d cytosines

HI' protons,

implying thereby

large ring current

chemical

shift of C14 Hl' proton

terminal

nucleotide

protons

loop out are expected in the helix.

It

la that the HI' p r o t o n of C8 resonates most u p f i e l d compa-

red to other cytosine helix e x p e r i e n c i n g

the ones which

shifts

that C8 unit stays

from the n e i g h b o u r i n g

is c o n s i s t e n t with the fact that,

and does not have any flanking purines.

loop out of the helix and do not experience

Similarly

it is seen in Figs.

appear most u p f i e l d compared an o b s e r v a t i o n which

1 and 2 that the H2',

The

C14 is a

that these

any ring current

H6 and H5 protons

to the r e s p e c t i v e protons

substantiates

G9.

The fact that HI'

of C6 and C7 also appear close to HI' of C14 implies

two units

in the

shifts. of C8

in the other cytosines,

that C8 base pairs with G9 on the opposite

strand and C6 and C7 loop out in both strands.

Further chemical

support

shifts

corresponding ion, Fig.

self c o m p l e m e n t a r y

6 shows the HI'

chemical

particular,

II has been o b t a i n e d by c o m p a r i n g

of HI', H2' and base protons

rum of the dodecamer. HI'

for structure

---

(H2', H2")

Comparing

shift patterns

dodecamer

in the 14-mer with those d-GAATTCGAATTC.

cross peak region of the COSY spect-

this with Fig.

are g e n e r a l l y

la,

similar

This implies that the two protons

mical e n v i r o n m e n t s

in the respective

requires

molecules

of the backbone

b o n d rotations.

some of these angles.

While the details

(i)

structure

In earlier papers

in COSS,

features

with HI' of

are in very similar

and c o n s e q u e n t l y

dihedral

cross peak intensities

ing the above

COSY and NOESY

che-

in the 14-mer,

in the m i s m a t c h

angles,

spectra can be used to fix structure

in a d e t a i l e d paper,

obtained

follow-

the important

angles are in the anti domain;

1230

and

shown how the

are outlined below: dihedral

region

sugar g e o m e t r i e s

(4-7), we have

of solution

strategies will be p u b l i s h e d

all the g l y c o s i d i c

that the

to C6 and C7 units only.

of the c o n f o r m a t i o n a l

the knowledge

glycosidic

it is o b s e r v e d

in the two cases and in

C8 in the 14-mer.

Finer details

in the

As an illustrat-

the Hl' of C6 in the 12-met has close c o r r e s p o n d e n c e

looping must be r e s t r i c t e d

the

VOI. 139, No. 3, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

~ C6

T(4,10)

©

c~

o

Tll

~ C6

A

¢J

08 G1

I.C)

&

I

~ T[4,10)

¢J

~

A3 ~ A 9

"I" GI ~ G 7

v

-~ G7

A2

AZ

© I'0 I

I

6.O

5"5

HI' Fig. 6.

(ii)

Portion of a 500 MHz COSY spectrum of d-GAATTCGAATTC. The region shows HI'--H2' and HI'--H2" cross peaks for all the 12 nucleotides. In C12, H2' and H2" are equivalent and thus only one cross peak is seen.

the conformations segment. observed

(iii)

of deoxyribose

It may be recalled in the B-DNA

region

the sugar conformations

rings

that similar

in the loop region

the loops are thus formed by changes with structural

variations

conformations

of GAATTCGAATTC

C6, C7 and G9 have a C3' exo geometry (iv)

are 01' endo in the -AATThave been

(8); are significantly

different.

while C8 has a C2' endogeometry; and in the sugar conformation

in the backbone

dihedral

coupled

angles.

ACKNOWLEDGEMENTS This work has been done on the 500 MHz FT-NMR National Facility supported by the Department of Science and Technology, Government of India.

REFERENCES I. 2. 3.

Govil, G. and Hosur, R.V. (1982) 'Conformation of Biological Molecules: New Results from NMR'. Springer Verlag, Heidelberg. Wang, A.H.J., Quiglay, G.J., Kolpak, R.J., van der Marel, G., van Boom, J.H. and Rich, A. (1981) Science 211, 171-176. Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaka, S., Itakura, K. and Dickerson, R.E. (1981) Proc. Nat. Acad. Sci. USA, 78, 2179-2183.

1231

Vol. 139, No. 3, 1986

4.

5. 6. 7. 8. 9. i0. Ii. 12. 13. 14. 15.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hosur, R.V., Ravikumar, M., Roy, K.B., Tan Zu-kun, Miles, H.T. AND Govil, G. (1985) 'Magnetic Resonance in Biology and Medicine' pp 243-260, Tata McGraw-Hill, New Delhi. Ravikumar, M., Hosur, R.V., Roy, K.B., Miles, H.T. and Govil, G. (1985) Biochemistry 24, 7703-7711. Hosur, R.V. (1986) Curr. Sci. 55, 597-605. Hosur, R.V., Ravikumar, M., Chary, K.V.R., Sheth, A., Govil, G., Tan Zukun and Miles, H.T., FEBS Lett. (in press)° Chary, K.V.R., Hosur, R.V., Govil, G., Tan Zu-kun and Miles, H.T. (1986) (to be published). Gronenborn, G. and Clore, C.M. (1985) Prog. NMR Spectrosc. 17, 1-32. Aue, W.P., Bartholdi, E. and Ernst, R.R. (1976) J. Chem. Phys. 64, 22292246. Hosur, R.V. Ravikumar, M. and Sheth, A. (1985) J. Magn. Reson. 65, 375381. Anil Kumar, Wagner, G., Ernst, R.R. and Wuthrich, K. (1980) Biochem. Biophys. Res. Commun. 96 1156-1163. Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol, 65, 499-560. Hare, D.R., W e m m e r , D.E., Chou, S.H., Drobny, G. and Reid, B. (1983) J. Mol. Biol. %7!, 319-336. Scheek, R.M., Russo, N., Boelens, R., Kaptein, R. and van Boom, J.H. (1983) J. Am. Chem. Soc. 105 , 2914-2916.

1232