Vol.

78,

No.

BIOCHEMICAL

3, 1977

THE

ROLE

IN

OF

DNA

REPLICATION

University

Biology

Received Summary Ealently exonuclease a significant

OF

Division,

Oak

August

29,1977

to earlier

polA480ex

(originally

exonuclease

C see Ray

(3)

with

DNA

Oak

Ridge,

Sciences,

Tennessee

coliphage

Ml3 into

and

Kornberg

and

5’

system

end

ssDNA, RF I DNA, in either

address: to whom by the

parental

37830

The site

sealed

the

role

synthesis

on the

Laboratory,

reprint

requests

should

Union

Carbide

Corporation

article, the a nonexclusive,

0 1977 by Academic of reproduction in any

Press, Inc. form reserved.

nature

of 2 X lo6 The

is synthesized

progeny

RF DNA to produce

of RNA

priming

template

University

mutant

E. coli -5’-

of the

daltons.

next

stage

of phage

synthesis,

the

RF I DNA

(3).

in Ml3

to generate

of Wisconsin,

form RF DNA

Madison,

DNA, DNA

template nascent

parental

DNA

3’

enzyme The

on RF DNA

of complementary-strand

viral-strand

DNA

temperature-sensitive

single-stranded DNA; RF DNA, replicative covalently closed RF DNA; RF II DNA, or both strands.

Biophysics

of this to retain

and

Ml 3 RF DNA in the 5’ + 3’ also contains

lethal

essential

DNA.

ssDNA

is subsequently

(4).

the ssDNA

pRF

progeny

established

at a fixed

circular

double-stranded

as RF II DNA

and

and

coli

conditional

I indicates

contains

finally

co-workers

of Escherichia

deficient

polymerase

is present

By acceptance Government the article.

of Biomedical

nonessentiality

containing

In both

*OP erated Administration.

Copyright All rights

polAex1)

of RF DNA;

for

at the

*Present

School

of o temperature-sensitive

a review].

Abbreviations: parental RF; discontinuities

Author

called

in an in vitro

primer

Mitrat

Laboratory,*

as to the

isolation

is converted

replication

t

recent

Filamentous

involves

thesis

conclusions

associated

infection,

DNA

DNA

conversion of both parentaland progeny-nascent open circular closed RF I is drastically reduced in an --E. coli mutant deficient associated with DNA polymerase I. The nascent progeny RF DNA proportion of fragments of smaller than unit length.

I, the

after

Sankar

Graduate

National

5’-EXONUCLEASE

REPLICATIVE-FORM

and

Ridge

COMMUNICATIONS

The

polymerase

2).

Ml3

Dasgupta*

Ridge

RESEARCH

I-ASSOCIATED

COLIPHAGE

of Tennessee-Oak

Contrary

(1,

BIOPHYSICAL

POLYMERASE

Santanu The

AND

RF syn-

utilizes

an RNA

RF II DNA. DNA; with

WI

RF

Sub-

pRF, one or more

53706.

be sent. for

the

Energy

Research

and

Development

publisher or recipient acknowledges the right of the U.S. royalty-free license in and to any copyright covering

1108 ISSN

0006-291X

BIOCHEMICAL

Vol. 78, No. 3, 1977

sequently

the

Schekman

et al.

out

by

well,

(4)

polymerase

In vivo

studies

involving

phage

DNA the

(5)

host

associated during

with both

presumably

of this DNA

parental

MATERIALS

dnoG -

and

thot

Thus contains

removal

of RNA

both that

in vivo

sealing and

and

has RNA

to produce

gap-filling

5’

replication

polymerose

RNA

7) showed

activity

to expect

thot

be carried

activity.

requires (6,

RF 1 DNA.

could

exonucleose

experiments

reasonoble

for

nascent

priming that

as

Ml3

RF

initiation

progeny

of RF DNA,

primers. is to present

I is indeed

3’

primer

RF DNA

which

report

involved

RF DNA

primers

ond

polymerose

progeny

it appears RNA

progeny

S-

the

Later

preliminary

RNA the

gop-filling

function,

polymerose

by removing showed

the

during

polymerase.

(8). also

purpose

that

suggested

RNA the

DNA,

DNA

I containing

synthesis

pRF

by

proposed

requires

The

recently

is replaced

DNA

replication

like

RNA

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

synthesis,

at the

exonucleose

5’ ends activity

evidence in the

in the

that

finol

sealing

absence

of nascent is required

the

DNA

5’-

exonuclease

of nascent

of progeny chains.

in Ml3

3’

ssDNA Chen

progeny

RF DNA synthesis, and

ssDNA

Roy

synthesis

AND METHODS

Bacterial strains and phoges - E. coli RS5052 (E. --coli polA480ex 5’- 3’ --exotsthy-su-F+), A spontaneous reveroriginally isolated by Konrad and LeKmanl), was agift of W. Masker. tont, RS5052rev, was isolated which, unlike the mutant, is viable at 43°C and resistant to methyl methanesulfonate. It behaves exactly like wild-type (K12 5274, --E. coli X1090 thy-su-). Ml3 gene 5 omber-mutant phage, am5H3 (Ml3 om5), and wild-type Ml3 phage -. were grfts of D. Pratt. Media and buffers - L-broth contained 10 g of Difco bactotryptone, extract, 10 g NoCl, and 1 g glucose/liter (supplemented with 20 mg/liter Supplemented M9 medium (7) contained 4 mg/liter of thymidine. Isolation of intracellular phage DNA - Log-phase oerotion and infected with phoge (100-200 phage/cell), on ethanol-phenol mixture (10) at -20°C to stop bacterial washed twice with 0.1 M NoCI, 50 mM TrisCl (pH 8.0)‘ 1 Om2 M KCN at 0°C and lysed OS described before (11). been described (11).

5 g of Difco of thymidine).

yeast

cultures of E. coli, grown under were mixedwxan equal volume of synthesis. The cells were then and 1 mM EDTA buffer containing The isolation of purified phage has

Ultracentrifugation procedures - Th e cellular lysotes were centrifuged in 5-20% sucrose gradient in 1 M NoCl, 20 mM TrisCi (pH 8.0), and 2 mM EDTA (11). Fractions of 0.7 ml were collected from the top, ond 0.1-0.2 ml aliquots were assayed for radioactivity. Two types of band sedimentation in alkaline sucrose (5-20% sucrose in 0.8 M NoCI, 0.2 M NoOH, For short cenrrifugotions, phage DNA (0.1 ml) was layered 2 mM EDTA) were carried out. on 4.9~ml gradients in Beckman SW50.1 swinging-bucket rotor tubes and spun at 45,000 rpm ot 20°C for 80 min. Fractions were collected from the bottom and assoyed for rodiooctivity. For extended centrifugations, DNA samples (0.2-0.4 ml) were spun in 10.5-ml gradients containing 1 ml 60% CsCI, and 60% sucrose (w/w) cushion in o Beckman SW41 swingingbucket rotor at 38,000 rpm for 12 h at 5°C. The method of radioactivity measurement and the chemicals used have been described (1 I ).

1109

(9)

Vol.

78,

No.

3, 1977

BIOCHEMICAL

rA RFII

AND

RF1

RESEARCH

B

+ I

I!

BIOPHYSICAL

30

i

. \

COMMUNICATIONS

.

I

olo

d-----O ./ .,a’

O

40

4

FRACTION NO

8 12 16 TIME (mtn)

20

RF synthesis in RS5052 and RS5052rev. Log-phase cultures of RS5052 and Figure 1 . Parental RS5052rev, grown at 33’C in supplemented L-broth, were shifted to 43°C 5 min before the addition of chloramphenicol (100 pg/ml). Ten min later, they were infected with 32P-labeled wild-type Ml3 (3 X 105 PFU/cpm) at a multiplicity of co. 200 and maintained at 43’C. At different times, lo-ml aliquots were taken and mixed wx ethanol-phenol, washed, lysed, and subjected to band sedimentation in sucrose gradient as described in Materials and Methods. Sedimentation was from (A) Sedimentation profile of intracellular DNA 7 min after infection. left to right. The positions of RF II, RF I DNAs, and unoltered phage (cp) are indicated. (B) The ratio of RF I to RF II in the cultures as a function of time after infection is measured from the total radioactivity under the peaks. Symbols: O-O, RS5052; O-O, RS5052rev.

RESULTS

AND

Ml3 by the

DISCUSSION

parental

host

functions

exonuclease RS5052

and

with

remarkable

[32 PlM13

synthesized

sealing

of RF the

results primers our

The

postinfection,

newly

DNA

and

RF II into

II

and

DNA

support at the

experimental

undetectable

the

idea

Since

5’ ends

of DNA

conditions, in RS5052

2,

4) that

the

(2),

the

5’+

investigated synthesis

(to

prevent

revertant 5’-

3’

exonuclease

and

the

was

seen

cpm)

associated

did

out

not

with occur,

albeit

a

to convert in the

(4)

5’ end,

in the

DNA

increase

ability

et al.

that

3’

However,

in their

role

point

5’-

gradient

revertant.

at the

entirely

protein

is involved

primer

out

by infecting

by sucrose

of Schekman

of RF II to RF I does

1110

DNA

has a vital we should

of the

phage-specific

exonuclease

studies

role

at 43’C

of parental

mutant

is carried

the

pRF

pRF has an RNA

3’ exonuclease

conversion

the

in vitro

However,

chains.

pRF synthesis

(1 .4 to 1 .9 X lo4

the the

the

in Ml3

the

in and

DNA

fate

RF DNA

Evidently

molecules.

(1,

the

in

mutant

RF I.

complementary-strand

I in Ml3

comparable

the

We

chloramphenicol

label

was

-Ml3

(3).

followed

total

between

polA480ex

polymerase with

phage

1).

difference

E. coli

and -in vitro

pretreated

(Fig.

7 min

in

-in vivo

with

RS5052rev

sedimentation

that

both

associated

synthesis)

after

RF synthesis

indicated our

removal even

in vivo of RNA

though,

polymerase at a slow

under I is rate

Vol.

78,

No.

BIOCHEMICAL

3, 1977

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

t

0

FRACTION NO

Figure 2. M9 medium multiplicity (20 @/ml) (A) and (8): RS5052rev in alkaline in the text. n;yely, C, 1 PlM13

0

60 120 180 PULSE LENGTH lsecl

3

Progeny RF synthesis in RS5052 and RS5052rev. Log-phase cultures in supplemented grown at 33’C were shifted to 43°C 10 min before infection with Ml3 am5 at a of 50-100. Ten min later, the cultures were labeled with E3Hlthymidine addition of the poison, for 1 min before and then processed as described in Fig. Band sedimentation of cell lysate in neutral sucrose gradient of RS5052 and respectively. Other details are as in Fig. 1. (C) and (D): Band sedimentation sucrose of pooled RF DNA from gradients in (A) and (B) respectively, as described Sedimentation was from right to left. The positions of RF I DNA and ssDNA: circular; L, linear; and F, fragments arising from RF II DNA are indicated. ssDNA is used as the internal marker.

1.

Figure 3. Conversion of RF II to RF I DNA. Log-phase cultures in supplemented M9 medium of wild-type E. coli X1090, and RS5052 were infected with Ml3 am5 at 33°C. -7 Aliquots of cultures were shrfted to 42°C 10 min after infection and then se labeled for different times with 20 &i/ml C3Hlthymidine. Total RF DNA was isolated as described in Fig. 2. The RF I/RF II ratio was determined for each sample by a short ultracentrifugation in alkaline sucrose as described in Materials and Methods. Symbols: O-O, Xl 090 at 42°C; A-A, RS5052 at 33°C; RS5052 at 42°C.

A- A,

(Fig.

1B).

of another

This 5’

III in --E. coli Progeny can

be studied

DNA with in the

synthesis

could

exonuclease (12)

be such

RF DNA

synthesis

exclusively

either The

in E. coli

in RS5052

(Fig.

2A,

between

of a trace 5’-3’

of this

exonuclease

enzyme

activity

associated

with

in the DNA

mutant

or

polymerase

an enzyme.

in a host

at 43°C

II ratio

result

activity.

may

C3Hlthymidine RF I/RF

be the

infected

and

B).

polA480ex with

RS5052rev

It is evident

RS5052

and

Ml3

Th e synthesis am5

(3).

by pulse

labeling

that

again

RS5052rev.

1111

-

here

of Ml3

We followed Ml3 there

progeny the

am5-infected is a significant

RF DNA progeny cultures difference

RF

Vol.

78,

No.

The pooled

structure

total

where

of these

RF DNA

that

proportion

in contrast as shorter

than

of 5’-

unit-length

DNA

showed

that

at the

permissive

These

results

activity

gradients

similar

sucrose

to separate the

ratio

sealing,

the

pulse version

that,

RF I from

deficiency

of the

than

of 5’-

compared

with These

its 5’-

that

have

RF DNA

recently

ribonucleotides. (unpublished

found

pulse

that

not

with

DNA

RF II into

33 and

and even

neutral

sucrose in alkaline

42’C,

(2)

after

a 30-set the

although 42’C

con-

the

total

was only

showed

I even

that

subsequent

in RS5052,

33 and

polymerase

RS5052

It is evident

where,

et al.

and

centrifugation

Methods.

at both

is

X1090

from

In contrast,

Uyemura

shown).

shown).

3’ exonuclease

--E. coli

--E. coli

in RF DNA

not

of nascent

short

at both

maintained

(data

of RF synthesis

as RF I.

and

restrictive

an acute

at 30°C

in polA480ex

type. with

the

the RNA

includes

In addition,

label

-am5

RF DNA

and

effect

depressed

at the

of RF I to RF 11 DNA

subsequent

in Materials

present

Ml3

am5-infected -

in wild-type

associated

consistent

role

B and

thymidine

labeled

the 5’-

ratio

into

unlabeled

conversion

the

the

fragments

was shifted

by pooling

combined

was

pulse with

in the

of Ml3

RF II is present

shown).

RF DNA,

where

2A,

was remarkably

in removing

This

Fig.

was

infected

experiment

efficient

in the wild are

3,

labeling

the

X1090 (data

in

3’ exonuclease, RF DNA.

in

during

that

in Fig.

in this

3’ exonuclease

results

role

II reflects

incorporation

lower

an important

in

in RS5052,

Thus,

with

not

temperature

parental

of pulse

was extremely

two-thirds

the

the

seen

(data

RF DNA were

RF II as described

of RF II to RF I DNA

slightly

as with

described

of RF I to RF

13H]thymidine

before

was quantitated

sealing

label,

for

clearly

to those

Ml3

cultures

of time

II ratio

when

label

of the

chase

in RS5052

RS5052rev

I plays

is more

as a function

RF I/RF

while

suggest

sealing

involving

occur

pulse

as again

Also

intermediates).

retarded

sucrose,

It is obvious

in RS5052.

of the

of

RF 11 sediment

D).

that

sedimentation

B) in alkaline

denatured

replication

experiments

band

2A,

2C,

than

fraction

causes

10 min

from (Fig.

higher

(presumably

obtained

and

polymerase

This

measured

also

in Fig.

DNA

a large

apparently

temperature strongly

RF I DNA.

RS5052rev,

of RF I does

were

by extended

strands

is much

However,

synthesis

single

unit-length

fragments

RS5052

of DNA

while

than

analyzed

27 of gradients

RS5052rev

in

strands.

after

was further

cushion

3’ exonuclease

results

temperature

strands

shorter

situation

a slow

Similar

Ml3

and

unit-length

deficiency

The

the

of RF I in

to the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

16 through

into

unit-length the

RF DNAs

(fractions

RF I sediments

circular,

We

BIOCHEMICAL

3, 1977

the

primers

sealing

nascent the

proposed

fragments

vital and

role

gap-filling

of discontinuous

RF II DNA contain

results).

1112

contains sequences

of DNA

polymerase

in both

parental

fragments multiple of both

I, and

in nascent gaps virus

and and

along

RF

a stretch

with

progeny

II DNA. of

complementary

Vol.

78,

No.

BIOCHEMICAL

3, 1977

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

ACKNOWLEDGMENT SD was Division

a postdoctoral

of Oak

Ridge

investigator

National

supported

Laboratory

by subcontract

to The

University

No.

3322

from

the

Biology

of Tennessee.

REFERENCES 1 . Konrad, E. B., and Lehman, 2048-4051. D., Eichler, D. 2. Uyemura, 4085 -4089 .

I. C.,

R.

(1974)

Proc.

and

Lehman,

I.

R.

Natl.

Acad.

Sci.

U.S.A.,

-’71

J.

Bioi.

Chem.,

-’251

(1976)

6. 7. 8. 9. 10.

(1977) In Comprehensive Virology (i-i. Fraenkel-Conrat and R. R. Wagner, Ray, D. S. eds.), Vol. 7, pp. 105-178. Plenum Press, New York. Schekman, R., Weiner, A., and Kornberg, A. (1974) Science, 186, 987-993. Brutlag, D., Schekman, R., and Kornberg, A. (1971) Proc. NatrAcad. Sci. U.S.A., 68, 2826-2829. Ey, D. S., Dueber, J., and Suggs, S. (1975) J. Virol., 16, 348-355. Dasgupta, S., and Mitra, S. (1976) Eur. J. Biochem., 67,27-51. Bouche, J. P., Zechel, K., and Kornberg, A. (1975) J. Biol. Chem., 250, 5995-6001. Chen, T. C., and Ray, D. S. (1976) J. Mol. Biol., 106, 589-604. (1975) In DNA Replication, Methods in Molecular Biology (R. B. Wickner, Okazaki, R.

11. 12.

ed.), Vol. Mitra, S., Livingston,

3. 4. 5.

7, pp. l-32. and Stallions, D. M., and

Marcel-Dekker, D. R. (1976). Richardson, C.

1113

C.

New York. Eur. J. Biochem., (1975) J. Biol.

67, 37-45. seem. -’250

470-478.