Isolation and characterization of naturally occurring hairpin structures in single-stranded DNA of coliphage M13

Vol.

79,

No.

4, 1977

BIOCHEMICAL

ISOLATION

AND

HAIRPIN

IN

Biology

November

BIOPHYSICAL

CHARACTERIZATION

STRUCTURES

OF

K.

Division, Oak

Niyogi Oak Ridge,

RESEARCH

DNA

and

COMMUNICATIONS

NATURALLY

SINGLE-STRANDED

Saiil

Received

AND

Sankar

OCCURRING

OF

COLIPHAGE

M13*

Mitra

Ridge National Laboratory, Tennessee 37830

4,1977

Summary -With precise conditions of digestion with single-strand-specific nucleases, m endonuclease Sl of Aspergillus oryzae and exonuclease I of Escherichia coli, nuclease-resistant DNA cores can be obtained reproducibly from single-stranded Ml3 DNA. The DNA cores are composed almost exclusively of two sizes (60 and 44 nucleoThese have high (G +C)-contents relative to that of intact Ml3 DNA, and tides long). The resistance of these fragments to arise from restricted regions of the Ml3 genome. single-strand-specific nucleases and their nondenaturability strongly suggest the presence of double-stranded segments in these core pieces. That the core pieces are only partially double-stranded is shown by their lack of complete base complementarity and their pattern of elution from hydroxyapatite.

The

genome

of filamentous

of 2 megadaltons digestion

consisting

with

Escherichia

the coli

ds-like

DNA 1 .5%

of the

a high

(G

+ C)-content,

.

ments?

Moreover,

unique

total

rapidly

remained

For

region

of the

action

endonuclease

in the

about

it renatured

solely

concomitant

and

core

coliphages

a simple

example,

of the

no data

major

had

were

fd.

an average

fd genome.

Bartok

The

chain

(2).

presented

et al.

core

and

However,

from

of one these

isolated

ssDNA

After

limit-

exonuclease

I of

(2)

a

obtained

fd DNA

of about

comprised

40 nucleotides,

ds-like

several

as to whether (3)

namely,

length

composed

et al.

is a circular (1).

Schaller

distribution,

preparation

etc.)

nucleases, crassa,

pyrimidine

the

fd,

deoxynucleotides

of ss-specific

denaturation was

M13,

of bacteriophage and

following

four

of Neurospora

ssDNA DNA

(e.g.,

spectral important

properties; questions

or several

distinct

fragments

arose

a nuclease-resistant

fragfrom

a

dsDNA

Abbreviations: ss, single-stranded; ds, double-stranded; RF DNA, replicative form DNA; endo R*HpaII, Haemophilus parainfluenzae restriction endonuclease; endo R. Hind, Haemophilus influenzae restriction endonuclease; EGTA, ethylenebis(oxyethylene nitrilo)tetraacetate; TCA, trichloroacetic acid; SDS, sodium dodecyl sulfate. By acceptance of this article, the publisher U.S. Government to retain a nonexclusive, covering the article. *Research Corporation.

Copyright All rights

supported

by the

0 1977 by Academic of reproduction in any

Press,

form

Department

Inc. reserved.

or recipient royalty-free

of Energy

under

acknowledges the right of the license in and to any copyright

contract

with

the

Union

Carbide

1037 ISSN

0006-291

A’

Vol. 79, No. 4, 1977

fraction

(2%

with

the

of total

ss-specific

denaturable large

authors

ssDNA two

because

may

act

the

exonucleases

two

size

that

of (pX174 of --N.

classes

DNA

(32

their

the viral

DNA

cmssa.

location

after

treatment,

Most

of this

48 nucleotides

of larger

present

RESEARCH COMMUNICATIONS

phage

and

fragments

” was also

suggested

AND BIOPHYSICAL

ssDNA

and

“tails,

cores

concluded

In view

have

endo-

16 base

also

from

contained

with

of the

containing

DNA)

of heterogeneous

pieces

analysis The

and

amount

core

BIOCHEMICAL

size,

in their

contains

one

DNA

long). presumably

non-.

However,

a

up of the

Pyrimidine-tract

regions

hairpin

was

made

preparation.

at specific

in succession,

in the

of 24 base

viral

DNA.

pairs

and

two

pairs.

of the of the

uncertainties intriguing

as specific

recognition

reinvestigated

the

isolation

in the possibility sites

methods that

for

and

such

enzymes

of isolating hairpin like

chamcterization

discrete

hairpin

structures

--E. coli of such

RNA

regions,

in an ssDNA polymemse

structures

and

genome (4),

in ss Ml3

we phage

DNA.

MATERIALS

DM2

Column cellulose

AND

METHODS

materials - Hydroxyapatite was a product of BioRad.

was

prepared

according

to Tiselius

et al.

(5).

DNA substrates - Bacteriophage Ml 3, labeled with 32P, was purified through two CsCl gradient centrifugations, and the DNA was isolated by phenol-SDS extraction following standard methods (6), then dialyzed against and stored in 0.01 M Tris (pH 7.8) buffer. 3H-labeled Ml3 RF1 DNA was obtained as described earlier (7). Calf thymus DNA was purchased from Worthington Biochemical Corporation. Enzymes - Pancreatic DNase (electrophoretically pure) and snake venom phosphodiestemse were purchased from Worthington Biochemical Corporation. The latter was The purification and digestion conditions with further purified according to Keller (8). Sl endonuclease (9) and E. coli exonuclease I (10) have already been described (6). Endo RaHpaII was purcha&l-&m New England Biolabs. Exonuclease VII of --E. coli was a gift of. J. W. Chase (11). of Ml3 DNA coresThe reaction mixture (2.0 ml) containing 30 mM H 4.6), 75 mM NaCl, 0.5 mM ZnCl2, 100 pg of 32P-labeled ss Ml3 and Sl endonuclease (300-500 units) was incubated at 30°C. DNA (3-6 X 1 $ cpm/pg) At various times, aliquots were taken out, to each of which 100 pg calf-thymus DNA and The precipitated DNA was collected on a Whatman GF/C TCA (to 5%) were added. disc, washed successively with 5% TCA containing 0.01 M pyrophosphate and with 70% The discs were then dried and counted for radioactivity in a toluene-based ethanol. scintillation solvent. After the desired extent of digestion, EGTA was added to 2 mM, followed by the addition of 4 ml of 100 mM No-glycinate (pH 9.5) containing 10 mM MgC12 and 7 mM 2-mercaptoethanol . Further incubation at 30°C was carried out with EDTA, NaCl, and 4-6 units of exonuclease I (10) till limit-digestion was achieved. sodium

SDS with layer

Isolation acetate

were then added phenol (saturated with 3 volumes

to 20 mM, 0.5 M, and 0.5% respectively. with 50 mM Na2B407), DNA was precipitated of ethanol at -2O’C, collected by centrifugation

1038

After

two extmctions from the aqueous (12,000 X g- for

Vol.

79,

No.

4, 1977

BIOCHEMICAL

0

K)

20

AND

30

BIOPHYSICAL

0 lo FRACTKYN NO.

RESEARCH

20

30

COMMUNICATIONS

40

Figure 1, Polyacrylamide gel electrophoretic patterns of Ml3 DNA cores obtained different degrees of predigestion with Sl endonuclease, followed by limit-digestion exonuclease I. A, B, C, and D correspond to 35%, 50%, 70%, and 80% digestion, respectively, with Sl endonuclease. The conditions of electrophoresis are described under Materials and Methods. The arrow indicates the position of bromophenol-blue dye marker.

30 min), centrated

dissolved in and extensively dialyzed by evaporation at room temperature

against 2 mM Tris-Cl before electrophoresis.

(pH

8.0),

and

after with

con-

Polyacrylamide gel electrophoresis -The routine isolation of core pieces was carried out by electrophoresing DNA in tubes (10 cm X 0.3 cm2) containing polyacrylamide (12% acrylamide, 0.2% bisacrylamide) in 40 mM Tris-acetate (pi-l 8.2), 20 mM Na-acetate, 1 mM EDTA, and 0.1% SDS, at a constant current of 4 mA per tube. The gels were sliced in 3-mm sections, soaked overnight in 2 ml of 20 mM Tris-Cl (pH 9.3), 2 mM EDTA, and monitored for radioactivity by Cerenkov radiation. Endo RmHpaII fragments of Ml3 RF DNA were discontinuous ~yacrylamide gels according to van DNA fragments were located on the gel by staining by soaking in 2 ml of 0.02 M Tris (pH 9.3) containing The according

size of DNA was determined to Maniatis et al. (14).

5S rRNA (120 nucleotides, R*Hlnd restriction fragments

in denaturing Th e calibration

ref. 15), fMet tRNA of (pX174 RF DNA,

separated by gel electrophoresis in den Hondel and Schoenmakers (12). with ethidium bromide (13) and eluted 2 mM EDTA. polyacrylamide of the gel was

from --E. coli and marker-dye

gels carried

in 7 M urea out with mouse

(77 nucleotides; xylene cyanol

ref. 15), FF (14).

endo

Hydroxyapati te chromatography -The samples were applied to small hydroxyapatite columns (1 ml bed vol). DNA was eluted by a 50-ml linear gradient of 0.005 M to 0.3 M No-phosphate (pH 6.8) containing 0.1 M KCl. One-ml fractions were collected. diluted to 3.0 ml with water, and counted for Cerenkov radiation. Nucleotide deoxynucleotides

composition analysis-32P-labeled DNA fragments by the consecutive action of pancreatic DNase and

1039

were digested snake venom

to 5’phos-

Vol.

79,

No. 4., 1977

BIOCHEMICAL

0

IO

20

AND

30

40

BIOPHYSICAL

0 FRACTION

lo No.

RESEARCH

20

30

COMMUNICATIONS

40

50

Polyacrylamide gel electrophoretic patterns of DNA core fragments I (A) Figure 2. and II (B) under denaturing conditions. Electrophoresis conditions (14) are described under Materials and Methods. XC and BB indicate positions of marker dyes xylene-cyanol FF and bromophenol blue, respectively.

phodiesterase BioGel DM2

as described by Fujimura column chromatography,

(16). The also according

nucleotides were then to Fujimura (16).

separated

by

RESULTS Effect

of nuclease

electrophoresis with

Sl

longer

patterns

pieces

tai Is at the until

the

(Fig.

Sl

pattern the

by the

stopping

fragments

with

Sl

were

essentially

(results

Lengths were

DNA

shown

shown).

The

the

3’ or 5’ end.

was

carried

out

was incubated

1 D from

VII,

are

of longer

completion,

has been

5’ ends

of ssDNA,

Ml 3 DNA

partially

the

reproducible

in a two-step

procedure,

simultaneously

with

Fig.

1 D,

absence

results

when

5’ end

VII

in

ss

material

exonuclease

shown

with

heterodisperse

80%

3’ and

that

of ssDNA)

because

profiles

both

indicating

Highly

It appears

end

ss tai Is at the

attacks

fragments

3’

of digestion

of nuclease-resistant

with

in Fig.

I.

to at least

resistant

which

the

The

out peaks

Limit-digestion

to exonuclease

resistant

the

not

not

from

extents

presumably I.

major

VII,

(17).

to those

(data

digestion

when

two arise

for

endonuclease,

is carried

with pieces

regions

at either

nuclease

longer

similar

nuclease

of ss tails

endonuclease

polyacrylamide-gel

exonuclease

I (specific

Sl

the

different

with

to exonuclease

use of exonuclease

at dsDNA

sized

resistant

1 shows after

limit-digestion

with

is obtained,

That

confirmed

II,

with

digestion

1 D).

than

are

Figure

obtained

to exonuclease

digestion

that

-

cores

by

resistant

of prior

5’ end

a consistent

DNA

followed

remain

extents

conditions

of Ml3

endonuclease,

smaller

digestion

produced

discrete-

predigested on the other

hand,

of substantial

were

as described Sl endonuclease

obtained

when

above, and

amounts the

rather

exonuclease

I

shown). of the

denatured

core

pieces

by heating

-The in 98%

two

peaks,

formamide

1040

shown

in

in o boiling

Fig. woter

1 D and bath

designated and

analyzed

1 and by

Vol.

79,

No.

4, 1977

BIOCHEMICAL

AND

BIGPHYSICAL

TABLE Base

Composition

of the

RESEARCH

COMMUNICATIONS

I

Ml3

DNA

Core

Fragments

The digestion of the DNA samples and separation nucleotides are described in Materials and Methods. in parentheses were published by Salivar et al. (18). Percent

of the 5’ The numbers

nucleotide

Nucleotide Virion

Core

II

31 15 22

25 18 29

tGG+C)

41 (40.9) 21 (21.1)

63 32

53 28

showed

component

a major

electrophoresis

under

of about

denaturing

conditions

60 nucleotides,

while

(14;

II had

and

see Fig.

a major

2).

component

1 of

44 nucleotides. Base

show

that

lished

composition while

values

contents

that

hence

of the

the (18)

agreement

T,

a partial

M KCI

40-50

nucleotide

When

with

Both

DNA

sufficient

would

these

fragments

cores

are

and

region,

nicked

0 few

minutes.

1 and

II even

DNA

Figure

at about

3 shows

0.12

and Similar

after

at 0.10

the

phosphate

at the

base

elution

with

(G

The

pub-

+ C)-

lack

of

complementarity,

profiles

of I and

buffer,

even

dsDNA

fragment

M phosphate (20).

This

rather, end,

have

I)

fragments.

at 0.20

base-paired;

again

them

are

presence

of about

buffer

ss regions

to render

in the

II on

(19,

20).

suggests

that

interspersed

resistant

to Sl

I. of the

core

to prolonged

denatured resistance

denaturation

partial

II (Table DNA

DNA.

M phosphate

to elute

viral

former,

A completely

be expected elute

intact

virion

only core

of I and

the

of intact

in the

particularly

nature

I and II are resistant

whereas

those

suggests

-

of the

especially

hand,

not extensively

exonuclease

Double-stranded ments

eluted

pairs

duplex

endonuclease

are

II,

than

structure

compositions

composition

I and

other

base

(and also at low temperatures).

denatured, Ml3

base

chromatography

te .

of 0.1

the both

on the

-The

pieces

higher

hairpin

Hydroxyapatite hydroxyapati

in

considerably

of A and

only

core

is remarkable,

are

equivalence

I,

I

20 (19.8) 24 (23.3) 35 (35.8)

gel

the

Core

C A T

polyacryiamide

about

DNA

Ml3

incubation RF DNA

to Sl treatment

fragments

with (21)

endonuclease involving

1041

- Figure

4 shows

Sl

endonuclease

is almost

completely

and heating

exonuclease and

quick

that

both and

core

exonuclease

digested I is shown cooling.

frag-

within by These

both

Vol.

79,

No.

1977

4,

BIOCHEMICAL

16-

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

z /‘-

/

/

/

FRACTKIN

Figure 3. described

and

other

Hydroxyapatite in Materials

results

(to

chromatography Methods.

and

be published)

/

/

/

that

2 -0.2

g r

No

of DNA

suggest

9 ::

--0.3

/ /

both

core

fragments

core

fragments

genome-I

and

I (A)

I and

and

II (B),

as

II possess

ds

tested

hybrid-

regions. Location ization

of the

to endo

immobilized hybridizes ments

R.HpoII

fragments

RF1 DNA

I hybridizes

to fragments

in selected

Ml3

of Ml3

filters.

efficiently

located

in the

fragments

on membrane most

are

core

most

F and

regions

of the

hairpin

regions

(12)

I (to Ml3

that

II were

were

efficiently

for

denatured

and

to fragment

be published).

F,

Thus,

both

while core

II frag-

genome.

DISCUSSION To obtain conditions

for

well-defined the

digestion

molecular-weight eliminated

Smaller

presumably

VII

contaminating if digestion

pletion.

at the

in removing The

5’

that

of their

havior

on hydroxyapatite.

render

them

molecules

are

held

together

tures

to ss-specific

from

-HpaII-F

to further

composed

I arises

base

nucleases predominantly

and -HpaII-I.

with

confirms are

not

compositions hairpin of Sl

rather

denaturation from

than

is suggested

the

It is interesting

precise high-

be completely

to at least

80%

longer

action

com-

ss tails,

of exonuclease

conclusion.

duplexes, for

contain

endonuclease

The

above

perfect

needs The

leave

I.

(particularly

structures,

base-pairing,

is carried

the

regions

can

endonucleose

material

action

after

Sl

to exonuclease

the

of hairpin

by partial

endonuclease

one

nucleases.

electropherograms

resistant

ssDNA

Yet

resistant

DNA

are

in Ml3

Sl

manner,

ssDNA-specific

in the

digestion

contaminating

regions

with

ss-specific

of prior

the

hairpin

by the

end,

ssDNA material

extents

noncomplementarity

Core

of Ml3

in a reproducible

as is evident A and

T) and

sufficient

their

ds segments

and

exonuclease

two

distant

by the

from

I.

ss regions

resistance

the

beto

That

these

of the

of these

treatment. endo that

1042

R-HpaII-F in the

fragment presence

while of --E. coli

core

Ml3

struc-

II arises

DNA-binding

Vol.

79,

No.

4, 1977

BIOCHEMICAL

0

I to

0

’

AND

8 m

BIGPHYSICAL

RESEARCH

I IO 300 NUJBATITION TIME (mm)

COMMUNICATIONS

20

xl

The resistance of DNA core fragments I and II to ss-specific nucleases. Figure 4. (A) Resistance to Sl endonuclease. Reaction mixtures (025 ml) containing 30 mM No-acetate (pH 4.6), 75 mM NaCI, 0.5 mM ZnCl , 32P-labeled Ml3 DNA core (10,000 cpm), nicked and denatured 3H-labeled M?3 RF DNA (10,000 cpm), and 10 units of Sl endonuclease were incubated at 30%. 4O-pl aliquots were assayed at indicated Reaction times for acid-insoluble radioactivity. (6) Resistance to exonuclease I. mixtures (0.35 ml) containing 66 mM Na-glycinate (pH 9.5), 6.6 mM MgCI 5 mM s-mercaptoethanol, 32P-labeled Ml3 DNA core (10,000 cpm), nicked and 3’enatured and 1.2 units of exonuclease I were incubated H-labeled Ml3 RF DNA (10,000 cpm), at 30°C. 50-pl 0, Nicked and A, 32P-labeled

protein,

DNA

in Ml3

ssDNA

and

own

our

protein

comparing

were assayed 3H-labeled fraction II.

replication

is initiated

to produce

the

(to be published)

indeed

the -HpaII-F

aliquots denatured core

restricts fragment),

their

ssDNA

presented

regions

arise

binding

Cathrine

those

predominantly

from

for

their

We

technical

RNA are

polymemse 22).

results

indicate

the -HpaII-F

of Schaller

to specific

that,

hairpin

although they

are

Brenda

by

region

et al.

site(s), such

in Ml3

of the

no means

Underwood

above

identical.

and

assistance.

REFERENCES 1. 2. 3.

Schaller, H. (1969) Schaller, H., Voss, Bartok, K., Harbers, 93-l 05.

J. Mol. Biol. H., and Gucker, B., and Denhardt,

44, r

1043

435-444. (1969) J. D. T. (1975)

Mol. J.

Biol. Mol.

(23)

(namely, sites

regions both

I;

DNA-binding

characterizing

occurring

to Ms.

radioactivity. core fraction

of --E. coli

palymemse

fmgment,

grateful

from

Results

at present

naturally

are

for acid-insoluble 0, 3*P-labeled

attachment

of the

the -HpaII-F

authors

(4,

prior

of --E. coli

Preliminary

here.

Snyder

that

times (21);

RNA

form

ssDNA.

with

Acknowledgment-The Ms.

ds replicative

on MI3

properties

by --E. coli

indicate

the

at indicated Ml3 RF DNA

44, &I.

445-458. 99, -

and

Vol.

4. 5.

6. 7.

a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

79,

No.

4,

1977

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Geider, K., and Kornberg, A. (1974) J. Biol. Chem. 249, 3999-4005. Tiselius, A., Hjer&, S., and Levin, 0. (1956) Arch.Biochem. Biophys. -65, 132-155. Mitra, S., and Stallions, D. R. (1976) Eur. J. Biochem. 67, 37-45. Dasgupta, S., Allison, D. P., Snyder, C. E., and Mitra, S.-(1977) J. Biol. Chem . 252, 5917-5923. Keller, E. B. (1964) Biochem. Biophys. Res. Commun. 17, 412-415. Vogt, V. M. (1973) Eur. J. Biochem. 33, 192-200. Lehman, I. R. (1966) In Procedures in Nzleic Acid Research (G. L. Cantoni and D. R. Davies, Eds.), vol. 1, pp. 284-295. Harper 8, Row, New ‘fork. Chase, J. W., and Richardson, C. C. (1974) J. Bioi. Chem. 249, 4545-4552. van den Hondel, C. A., and Schoenmakers, J. G. G. (1975) Eur. J. Biochem. 53, 547-558. %arp, P. A., Sugden, B., and Sambrook, J. (1973) Biochemistry -12, 30553063. Maniatis, T., Jeffrey, A., and van de Sande, H. (1975) Biochemistry 2, 3787-3794. Barrell, 8. G., and Clark, B. F. C. (1974) In Handbook of Nucleic Acid Sequences, Joynson-Bruwers Ltd., Oxford. pp. 70 and 32. Fujimura, R. K. (1970) Anal. Biochem. 36, 62-71 . Chase, J. W., and Richardson, C. C. (1973 J. Biol. Chem. 249, 4553-4561. Safivar, W. O., Tzagaloff, H., and Pratt, D. (1964) Virology-g, 359-371. Wilson, D. A., and Thomas, C. A., Jr. (1973) Biochim. Biophys. Acta 331, 333-340. Niyogi, S. K., and Underwood, B. H. (1975) J. Mol. Biol. 94, 527-535. Allison, D. P., Ganesan, A. T., Olson, A. C., Snyder, C. M.>nd Mitra, 5. (1977) J. Virol., in press. Tabak, H. F., Griffith, J . , Geider, K., Schaller, H., and Kornberg, A. (1974) J. Biol. Chem. 249, 30493054. Schaller, H ., UhGnn, A., and Geider, K. (1976) Proc. Natl. Acad. Sci . U.S.A. 73, 49-53. -

1044