The rolling-circle replicative structure of a bacteriophage λ DNA

Vol.61,No.2,1974

BIOCHEMICAL

THE ROLLING-CIRCLE

REPLICATIVE

STRUCTURE OF A BACTERIOPHAGE h DNA

Seishi Biophysics Laboratory University of Wisconsin,

Received

September

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Takahashi and Biochemistry Department Madison, Wisconsin, 53706, U.S.A.

23,1974

SUMMARY: Intermediates of h DNA replication in the second half of the latent periodhave been isolated and investigated in the electron microscope. The isolated replicative structures were predominantly single-branched "rollingcircle" replicative forms. The long linear tails (concatemers) may be the precursor of mature A DNA. INTRODUCTION: gradient of the form

The finding

(1,2,3) latent

which

has suggested period

should

generates

found

system

(Ret

structure

int-)

can be produced

Sensitivity

to physical

(consistent

with

for

this

suggests

that

single-stranded observed

(6,7).

in the present

study

in a catenated

form while

absence

structure preserving

protected

DNA

deficient this

DEAE cellulose have been reported sensitivity

of this

by the Ic gam gene product

linear

free

structure

original

(the

of recombination.

regions)

can reproduce the

that

to benzoylated

with

half

replicative

length

the argument

The replicative is compatible

second

circles.

The -recBC-nuclease

normally

sucrose

in a recombination

in the

and binding

round

the monomer

found

form contains

regions

replicative

from parental than

in the

first

of single-stranded

is

replicative

form

the

strengthening

(5).

which

rolling-circle

from

by replication

structure

this

replicative

is- longer

(4),

shearing

structure,

h DNA in an alkaline

was also

the existence

replicating

replicating

circles

particles)

red-,

the

be different

DNA which

in phage A-,

that

progeny

Fast-sedimenting length

of fast-sedimenting

these

ends or extended ("rolling-circle") properties.

more than circular

an entire

The genome

template.

preculture was grown in D-medium (9) at MATERIALS AND METHODS: An E. coli 37°C. The preculture was inoculated into 20 ml of fresh D-medium and the cells The cells were centrifuged, and resuspended in 1 ml of grown to A5gO = 0.70. Tris-MgS04, 0.01 M each in D20. After starving the cells for 15 min at 4"C,

Vol.

61, No.

2, 1974

BIOCHEMICAL

AND

658

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

BIOCHEMICAL

Vol.61,No.2,1974

C3H]-labelled and incubated The infected at 37°C.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A phage (in D20) was added at a multiplicity of infection of 10, at 4°C for 20 min for phage absorption (D20 freezes at 3.8"C). cells were diluted with 20 ml of prewarmed D-medium, and incubated

DNA replication was terminated by pouring the cells into 20 ml of an ice cold solution containing 0.01 M-KCN, 0.15 M-NaCl and 0.015 M-sodium citrate. Lysozyme-pronase treated lysates were made up to the density of 1.67 with CsCl and centrifuged (Ti-60 rotor, 30,000 rev/min for 3 days at 7'C). The fractions containing DNA of density between half heavy and heavy were used for preparation of electron microscopic specimens. Molecules were photographed on 35 mm film and measured with a curve length integrating device. Data computation was as described previously (9). RESULTS AND DISCUSSION: found

in fractions

gradient

exhibited

branched

theta

This

located

(8)

which

between

light

molecules

structure

is

generates

question

is whether

Table

Summary of electron

and hybrid

similar

to represent

a progeny the

circle

second

round

microscopic

density

from

consisting

found the

of double-

in E. coli

(8,9,10).

first

round

of h DNA

a parental

circle.

An

k DNA replication

proceeds

in

observation.

between light and heavy light or between Figure 1 were prepared by standard neutral are denatured with high temperature Form. No. of doublebranched

No. of molecules scanned

67

163

230

126

2

128

30 min

s/e

(X)

29.1 98.4 181

50 min

Plate

the molecules in a CsCl density

structure

to those

believed

DNA solutions in fractions located heavy light and heavy positions in spreading procedures (9). *Molecules (50°C for 30 min). **RF: Replicative No. of singleTime branched after infection 15 min

have shown that

replicating

type

important

1.

experiments

a characteristic

replicating

replication,

Previous

1.

Electron

(53)*

toI*

(53)*

168

10

178

micrograph

of a "rolling-circle"

(loo.o)* 94.4

h DNA.

Branch point is shown by a black arrow. Attached tail possesses several singleEnd of the tail is shown by white arrow. This molecule is stranded segments. 215% replicated (circular length is 17.7 micrometers and tail length is 38.1 micrometers).

659

Vol. 6 1, No. 2, 1974

the same manner different

BIOCHEMICAL

as the

process.

first

round

To answer

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of replication

this

question,

or whether we have carried

it

involves

out

a

the following

experiments: Three

cultures

phage

and incubated

Fifteen

minutes

found

in a half

grown

in heavy medium were

in heavy medium for

15 min,

after

infection,

about

heavy

position,

indicating

replicate

[Figure

between

the density

Observations

l-(a)]. of light

and half

infected

I)IL

[3H]-labelled

30 min and 50 min,

36% of the that

label

parental

heavy peaks

h

respectively.

in parental

DNA was

h DNA had started

on the replicating exhibited

molecules

to

located

predominantly

(71%)

(bl

(a) I””

with

i’

in

rL

IO00

(LL

(c) I“”

lHL

ILL

t ?

z P

500

30

20

IO

30

Figure E. coli T08/,1. extracted in the DNA are on the infection activity

1.

IO 30

20

FRACTION

IO

NUMBER

CsCl density-gradient centrifugation grown in a heavy medium, infected

with

of DNA from cells light [3H]-h.

which

W3102 (&, -su-) was grown in heavy medium at a cell densi [3H]-labelled h phage were added at a multiplicity of 10. and subjected to CsCl density-gradient centrifugation as methods. The positions where the heavy, the half heavy and located are indicated with the signs HH, HL and LL respecti density). 15 minutes after infection at 37Y (a), 30 minutes (b), and 50 minutes after infection (c). The recovery of was (a) 97%, (b) 93%, (c) 90%, respectively.

660

are

ty of 2 x DNA was described the light vely (based after radio-

V0l.61,N0.2,1974

theta the

(0)

type

previous

replicating

molecules minutes

the half

heavy position molecules

positions.

Almost

single-branched traction

delta

of this

kind

replication

after

infection

denser

position

(98%)

of these

replicating

example,

of the

attached

to their

denatured

sites

segments molecules

region

close

(about

18% from

tion

from

this

position

sites

circles

the

a region could

were

also

at

a

the DNA exMolecules

mechanism (11).

Fifty

was located

even minutes

at a still

single-branched

from

on the

to determine

h DNA are free

circles

they

are a

A DNA derived

from

a

denaturation

listed

and the

suggesting

to this

point.

Using

in

that

Figure

2.

round

these

preparation

the

the

segments of the

which

have longer

Out of 20 molecules, arrow)

located

at a

of h DNA replication

molecules

that

as

as a gauge

tail

by vertical

The fact during

extra

molecules

of the first

661

to recognize

to the correspondence

end (shown

end)

possible

pattern

X DNA, we have estimated

replicative

right

of linear

whether

cells.

according

origin

be due to breakage

infected

circle,

postulated

close

length

Linear

linear

Twenty

had their

a variable

map of A DNA is

are disposed

circular

(40%) to the

that samples.

a characteristic

purified

to the circles.

eight

and late

be involved).

from mature

cohesive

than

located

exhibited

the fact

denatured

denaturation

map derived

position

molecules

DNA label

possess

exhibited

A DNA in a DNA preparation

tail

which

DNA might

distinctive

denaturation

circles

molecules).

(E-. coli for

(9).

heavy and heavy

imprecisely

parental

have been partially

A phage,

progeny

by a rolling-circle

molecules

molecules

portion

This

the

with

85% of the structure

the half

the early

or terminated

l-(c)],

consistent

and we have observed

despite

completely

and replicating

A DNA or not

(9).

structure for

initiated

The replicating

mature

type

be copied

(94% of 178 replicating

branched

to replicate between

quite

circular the

fractions

[Figure

is

of infection,

l-(b)],

in the

(a)

were

10 minutes

[Figure

was identical

could

result

a double-branched

had started

all

procedure

This

after

infection

replicating

if

that, exhibited

after

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

structures.

observation

replicating Thirty

BIOCHEMICAL

not

all rather

started tails than

replicaended that

in they

Vol.61,No.

2,1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

15

1

16

z

1

3

ma..

1

I1

4 I6

5 19

6 1 6

3 IO 11 I2

I3

5

lb

1;

MICROMETERS

I4

Figure

2.

Electron structures

microscopic denaturation with a long tail.

maps of the 20 h replicative

The circles are artificially broken at the cohesive sites based on the denaturation maps and are normalized to 17.5 fl, the average length of mature phage A DNA. The attached tail segments have also been normalized. The denatured sites of the tail were then aligned with those on the circle. Branch point and free end are shown by vertical line and arrow respectively. Histogram shows the weight average of the circle with tail segment. The number of molecules used for this histogram was 53.

had started

in other

regions.

replication,

we observed

be explained

either

result

system

(RecA-B-

the extraction

predominantly

by a shift

of recombination

have observed

After

rolling-circle x int-,red-,gam-).

a single-branched

to a different

between

circular

molecules

of late

replication

and linear

stage

molecule.

This

process

or as a

molecules.

even in a recombination (Data

662

will

be published

of h DNA

However,

would

we

deficient elsewhere.)

These

BIOCHEMICAL

Vol.61,No.2,1974

results

indicate

DNA would (12).

that

be generated

Rolling-circle

direction. bidirectional

This

the molecules if

which

replication

molecules

have a longer

proceeded

observed

bidirectionality

replication

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

could

of the first

ACKNOWLEDGMENT: We acknowledge critical reading and suggestions Baldwin, W. Dove and A. Skalka

as in the

in the be related round

then

present

genome length

rolling-circle

study

rolled

to the mechanism

A model

in either of

of h DNA replication.

Dr. R. Inman for his encouragement. The on this manuscript contributed by Drs. R. are gratefully noted.

REFERENCES: 1.

$th,

2. 3.

Young, E. and Sinsheimer, R. (1968) J. Mol. Biol. 33, 49. Carter, B. J., Shaw, B. D. and Smith, M. G. (1969) Biochim. Biophys. Acta 195, 494. Skalka, A. (1971) In The Bacteriophage Lambda, ed. by A. D. Hershey, Cold Spr. Harb., N. Y., p. 535. Kiger, J. and Sinsheimer, R. (1969) J. Mol. Biol. 40, 467. Enquist, L. W. and Skalka, A. (1973) J. Mol. Biol. 75, 183. Unger, R. C. and Clark, A. J. (1973) J. Mol. Biol. 70, 539. J. and Fuke, M. (1968) Proc. Nat. Acad. Sci., U.S.A. Ogawa, T., Tomizawa, 6J, 861. ;;:E;, y. and Inman, R. B. (1970) J. Mol. Biol..a, 61. (1963) Cold Spr. Harb. Symp. Quant. Biol. 28, 43. Skalka: A:, Poonian, M. and Bartl, P. (1972) J. Mol. mol. 64, 541. G,;lb$, W. and Dressler, D. (1968) Cold Spr. Harb. Symp. Quant. Biol. -' *

4. Z: 7. 8. 9. if: 12.

M. G. and Skalka,

A.

(1966)

J.

663

Gen. Physiol.

46,

No. 6, Part

2,