Changes in the sedimentation characteristics of DNA due to methylmercuration

Vol. 40, No. 3, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

CHANGES IN THE SEDIMENTATION CHARACTERISTICS

OF DNA DUE

TO METHYLMERCURATION Dieter Department

of

W. Gruenwedel

Food Science

h Technology,

Davis,

Received

and Don S. Lu

California

University

of

California

95616

June 11, 1970 SUMMARY

The methylmercuric ion induced denaturation of DNA has been followed by determining the rate of sedimentation of native T7 DNA at neutral pH as a function of increasing CH3HgOH concentration in solvents of varying ionic strength. The denaturation and subsequent complexing reaction of DNA by CH3Hg+ appears to proceed in two steps: at first, there is a highly cooperative transition of the DNA from the native to the denatured state as indicated by an abrupt increase of ~by a factor of about 3. This is followed by a second sharp transition at higher CH3HgOH concentrations in which 2 increases additionally by a factor of 3 or more. The nature of this very rapidly sedimenting CH3HgDNA complex is unknown at present. Methylmercuric We have

studied

ions this

react

reaction

in an ultracentrifuge

measurements

are:

not

affect

the buoyant

is

reached

now binds the

beyond

buoyant

c) the

centration

denatured

DNA complex

DNA with

of sedimentation

the

more

to study

facts

very

about

density

buoyant

density

CH3HgOH does

CH 3HgOH concentration to denatured

dense

DNA which

and nonbuoyant;

amount

nonbuoyant

increase

b) in

the

of methylmercury at

the

same con-

DNA. the properties

the methylmercury-DNA

experiments.

the

a gradual

in the

becomes

native

of

transition

CH3HgOH produces increase

denaturation.

and by buoyant

a critical

becomes

CH3Hg-DNA complex

to learn

velocity

it

a gradual

of CH3HgOH as does

we decided

a cooperative

prior

DNA in Cs2S04 with

the DNA until

that

indicating

In an attempt

presented

is

(1)

The salient

of native of

there

of denatured

density,

bound;

titration

so much methylmercury

titration

DNA by causing

(2).

density

which

native

spectrophotometrically

measurements

a) the

with

Some results

below. 542

of

the

interaction of this

nonbuoyant with investigation

CH3Hgthe

help are

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 40, No. 3, 1970

MATERIALS AND METHODS T7 phage

were

centrifugation of

grown

and by banding

the phenol

extraction

The DNA was stored cacodylic 0.05

acid,

salt"

("high

Na2S04

+ 0.005

0.0025

g Na2S04 + 0.005

cacodylic g/ml

bulk

+ 0.16

24,000

determine

the

are

at

in 0.05

g Na2S04 were

+ 0.9 -M Cs2S04,

pH 6.8,

g/ml

if

acid

at

the

pH 6.8,

("low

bulk

-M

using

as a stabilizing g

was performed pH 6.8,

as in 0.0025 salt"

+ 0.005

20" C) or in 0.0025

g Cs2S04,

20" C) as well

(6).

performed

sedimentation

+ 0.45

the help

previously

experiments

experiments

of 0.5

self-generating

ug/50

in

("medium

salt'

M Na2S04 + 0.005

solution;

3

p = 1.044

uncorrected

solutions

the

The lamellar

described

of the above.

DNA concentrations. for

gradients

low concentrations

composition

routinely

was used

-s-values

to

contained

polydispersity

DNA solutions

bulk

in order

CH3HgOH) to 11 ug/50

increased

lamellar

C and

The method

solutions

of

Both

All

at 20"

E Ultracentrifuge.

of CH3HgOH) due to the

The ionic

identical

Model

(7-9).

~1 (at

performed

density

sedimentation

concentrations

DNA stock

were

rpm in a Beckman in

the DNA samples.

contained

g/ml

velocity

rate from

limiting

the

M cacodylic

rpm or 26,000

DNA ranging

of

either

pH 6.8,

g Cs2S04,

of band sedimentation

(at

as described

p = 1.250

acid,

with

20" C).

Sedimentation at

acid

by differential

T7 DNA was isolated

and purified

solution;

p = 1.127

(4).

the sedimentation

g cacodylic

acid

at

if

and purified

chloroform

JJ cacodylic

medium

solution;

(5)

at 4" C over

pH 6.8,

B (3)

in CsCl

method

-M Na2S04 + 0.005

bulk

on j$. __ coli

and lamellar , given

~1 of

was that solutions

in Svedberg

units,

solvent. RESULTS AND DISCUSSION

The addition T7 DNA does not critical (Fig.

of increasing alter

the rate

CH3HgOH concentration 1, curves

pertaining at pM = 3.5

amounts of

A, "high

[PM = - log

sedimentation

is reached,

A and B, and Fig.

to curve

of methylmercuric

salt"

(CH3HgOH)I.

2).

beyond

At the

bulk

of the

macromolecules s suddenly

particular

salt

the

increase

solution,

At pM = 3.3, 543

which

hydroxide

2 appears

to native until increases

concentration in s commences to reach

a plateau

a

BIOCHEMICAL

Vol. 40, No. 3, 1970

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

80

l

2

3

4

5

6

00

DM

s as a function Fig. 1. Plot of the rate of sedimentation pM = - log (CH3HgOH). Curve A: "high salt';- bulk solution. "medium salt" bulk solution (see MATERIALS AND METHODS).

of pM. Curve

B:

120 -

100 -

80 -4

60 -

40 -

20 -

o-

12)456\ PM

Fig. 2. Plot of the rate of pM = - log (CH3HgOH). Curve AND METHODS).

sedimentation C: "low salt"

544

5 as a function bulk solution

of pM. (see MATERIALS

BIOCHEMICAL

Vol. 40, No. 3, 1970

region rate

after

having

increased

of sedimentation It

a sufficiently sediments

with

pM < 3.5

yields

denatured until

strong

complexing

a rate

equal

DNA.

abruptly

are

in complete

buoyant

density

mercuric

ions

Curve

occurs

ionic

the

decrease

with

with

solution, "low

behavior

of T7 DNA exhibited

increase

in 2 of T7 DNA in the by a sudden

renewed

increase

PM-values

of

the various

It

is

-s is

in

the

that

the

of strand

flexibility separation

the

the

of 5 on pM at

solutions.

denaturation

salt"

in

solution,

as well

as "low

were in the aware

the salt" with

the

this of

reproducible

545

solution

a subsequently the g,

and thus

characteristics

we may have

increase the

radius

the macromolecules. and the

bulk

overlooked

of the

medium.

initial

lowers

at

initial

Although

sedimentation that

"medium

to increase

sedimentation

highly

the

proceeds

the sedimentation

bulk

of

Due to

reaction

5 starts to

and

DNA (1,2).

For instance,

salt"

results

of methyl-

CH3HgOH concentrations.

"high

CH3HgOH of

These

interaction

bulk

rate

differences

to associate

DNA.

sedimentation

curves

amount

to single-stranded

salt"

with

of our spectrophotometric

In contrast

"medium

CH3HgOH at

react

a large

dependence

"high

titration

since

point

2, show the

solution.

higher

in s in the

structure

enhances a result

still

DNA does not

methylmercurated

ultimately

we are nevertheless

reasonable

of the helical quently,

drop

of alkali

at which

was shown

in the

to us real

macromolecules,

corresponding

bulk

DNA that

to that

at pM = 3.8 whereas

decrease

of s at

seem to indicate the

salt"

yields

comparable

concentration,

s increases

cyanide,

by adding

of

results

C, Fig.

instance,

removal

CH HgOH concentrations. 3

in the

followed

it

ion

pM = 4.1

is

the

corresponding

counter

lower

reached,

for

the

2 = 170.

DNA whereas

native

denatured

1, and curve

in the

bulk

is

that

at pM = 3.0,

at pM = 2.6,

as sodium

at a rate

DNA leads

of the

such

this

in which

native

strength

at progressively salt"

from

yielding

studies

indeed,

of native

sediments

agreement

B, Fig.

the

agent

concentration

reaction

sharply;

of CH3HgOH at pM > 3.5,

to that

We conclude

a critical

thus

again

removal

a DNA that

However,

from 5 = 12 to 2 = 36.

increases

can be shown that

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

accompanying

in -s with of gyration

a breakdown and,

The subsequent decrease

consedrop

in molecular

in

BIOCHEMICAL

Vol. 40, No. 3, 1970

weight.

Indeed,

B, Fig. 36.0,

the

1) , s = 36.5, at

the

in

formaldehyde

studying (lo),

and by Zimmer helix-coil

easily

explained.

brought

about

guanosine

this

the

observed

solvent

by the sites

f are

of

and with

the

the

mo:e

However,

value

and cease

in detail:

species,

same time,

of bound

preferential

coefficient

acid

DNA (ll), induced

group.

and the bound yields set

of pM.

limit

of infinitely

ENnf

(II)] weight

the

(13-15). per

If mole

of bound where

water

M3 is

of water

Let

the

sites,

us conpolymer, of the

- V,P) [eqn. volume

mass

binding

diluted

= Ms(l

and

a high

to properties

specific

cation

less

thymidine has

to be a function

in moles

(01

and frictional

density

and relative

we define

with

rb -

of DNA nucleotide per mole unhydrous

DNA molecular

and M, the molecular

Similarly, + TM1 + rbMx) [eqn. (III)] where ii,, vl, and specific volumes of the polymer, the water

methylmercury

group.

ENnf

= M3(1

- v,p)

equal

to l/p.

If

of single-stranded

cation of all

number

solvation

is

saturation

partial

methylmercuric

+ rbMxvx)/(M3 respectively, the partial

Vl is

of

of

to the

this

and p, n and N are

and Avogadro's

vs = (M3c3 + LM1vl

(I)

the

cation

equation

weight,

the methylmercury

into

the presence

due to the mass increase

s may be related

by the

molecular

solvent

undoubtedly

after

the

coefficient species

the

at

of

(III)

DNA, s=

by Freifelder

denaturation

DNA since

(1,2).

weight

solvation

curve

denatured

obtained

alkaline

(cf.

CH3HgOH concentrations

is

of single-stranded

of

of

alkali

were

the methylmercuric

weight

are,

of

concerning

the higher

MS = M3 + TM1 + rbMx [eqn. the polymer, Ml the molecular

vX

the

increase

of

solvated

of moles r the

nucleotide,

the

sedimentation

of

at pM = 3.5

of T7 DNA in

investigations

of 5 at

volume

somewhat

coefficient

their

that

results

in studying

in

a limiting

M s' i s'

number

denaturation

addition

and sedimenting

viscosity

the heat

In part,

and a low specific

sider

with

Similar

increase

reach

measured

of DNA (12).

binding

2 should

identical

strength.

and Triebel

transition

coefficient

almost

by Studier

The renewed

the

is

same ionic

and Davison

where

sedimentation

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DNA is

Substitution

of eqns.

(II)

and

+ rbMx(l - vxp) [eqn. (IV)] if, at the it is further assumed that the frictional

unaffected

546

by methylmercuration,

eqn.

(IV)

BIOCHEMICAL

Vol. 40, No. 3, 1970

permits

an estimate

to denatured of

DNA.

T7 DNA), that

binding

obtain

from

eqn.

that

2 indeed

region

We set

(IV)

?

x

this

the

We are,

however,

at a loss

of the

cesium

is

by the

from

in

the

shows

the pM value of

all

at

the plateau

likelihood

mass increase

to explain

salt

2 [P + l/vl]~

the beginning

interpretation caused

CH3Hg-group

C, Fig.

1.5 when going at

of the

and rb = 0.5 [that is, we T and G; see ref. (2)] and

Curve

pM values

in g is

weight

e.g.,

of about

above

addition

= 216,

x

= 1.45.

to the

change

M

to two bases,

by a factor

RESEARCH COMMUNICATIONS

molecular

ml/g,

~(rb=0.5)/~(rb=O)

to us that

and that

= 0.19

only

separation

indicating

in 2 due to the

M3 = 441 (monomer

ml/g,

increases

mercuration.

increase

occurs

of strand

correct

the

v3 = 0.47

assume

the point

of

AND BIOPHYSICAL

due to methyl-

dramatic

increase

in -s

at pM < 3. Figs. salt"

bulk

rapidly the

1 and 2 show that solution

than

sedimenting

supporting

buoyant nonbuoyant

the

measurements

net

v1 = l/p experimental possible

the

the

formation

brought

mercurated

DNA.

that,

(2)

high addition

and that

results that

is

swill

presented

thus

sharp

rise

about

by the

in order

that

this

instance, medium

This

calf

"high

to produce

the

continues

thymus

containing

about high

if

in our DNA becomes 1.6 g Cs2S04( 2)

CH3HgOH concen-

or correspondingly to DNA causes

would

in the

Indeed,

at a sufficiently

CH3Hg-group

mean that

the

also

be a function

of r

far

seem to support

this

of 2 at pM < 3 reflects increase

trend

increased.

Cs2S04 concentrations, of the

is needed

solution

further for

that

of the polymer.

invalid

bulk We expect

we find

proposed

hydration is

salt"

(2 > 0) in a buoyancy

previously

activities,

in the

"low

concentration

and at sufficiently

water

CH3HgOH concentration

CH3Hg-DNA species.

at pM = 3.2

We have tration

in

electrolyte

density

a lower

the

in hydrophobic

an abrupt

decrease

approximation

[eqn.

(IV)].

notion.

onset

low

The It

is

of microgel

character

of

the methyl-

ACKNOWLEDGMENTS This Public

research

Health The authors

with

has been

supported

by grant

GM 16282

from

the

United

help

in providing

States

Service. would

like

to thank

Mr.

E. Low for

T7 bacteriophage. 547

his

them

Vol. 40, No. 3, 1970

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

D. W. Gruenwedel and N. Davidson, J. Mol. Biol., 21, 129 (1966) D. W. Gruenwedel and N. Davidson, Biopolymers, 2, 847 (1967) K. D. Lunan and R. L. Sinsheimer, Virology, 2, 455 (1956) C. A. Thomas, Jr. and J. Abelson, In "Procedures in Nucleic Acid Research", ed. by G. L. Cantoni and D. R. Davies, Harper & Row, Publishers, New York and London, 1966, pp. 553 J. D. Mandell and A. D. Hershey, Anal. Biochem., 1, 66 (1960) D. W. Gruenwedel and Chi-hsia Hsu, Biopolymers, 7, 557 (1969) J. Vinograd, R. Bruner, R. Kent, and J. Weigle, Proc. Nat. Acad. Sci., U.S., 9, 902 (1963) J. Vinograd and R. Bruner, Biopolymers, A, 131 (1966) J. Vinograd and R. Bruner, Biopolymers, 5, 157 (1966) D. Freifelder and P. F. Davison, Biophys. J., 3, 49 (1963) F. W. Studier, J. Mol. Biol., 2, 373 (1965) Ch. Zimmer and H. Triebel, Biopolymers, 8, 573 (1969) R. J. Goldberg, J. Phys. Chem., 57, 194 (1953). S. Katz and H. K, Schachman, Biochim. Biophys. Acta, 18, 28 (1955) R. Bruner and J. Vinograd, Biochim. Biophys. Acta, 108, 18 (1965)

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