BIOCHEMICAL

Vol. 70, No. 2, 1976

"Distinguishable

Department

RESEARCH COMMUNICATIONS

Steps in the Enzymatic Synthesis

of Bacteriophage

Ranjit

AND BIOPHYSICAL

$X174 Replicative

Form -In Vitro"

Ray, Daniel Capon and Malcolm Gefter

of Biology,

Massachusetts

Institute

of Technology

Cambridge, Massachusetts Received

March

12,1976 SUMMARY

form synthesis On the basis of kinetic evidence bacteriophage $X174 replicative The first stage reaction converts the DNA to an occurs in at least two stages. This "activated" form may be isolated activated form in the absence of DNA synthesis. and can be shown to have different properties than unreacted DNA. The second stage comprises the DNA synthetic reaction but also requiresthe dnaG protein. INTRODUCTION The conversion double-stranded,

--in vitro

replicative

of bacteriophage form requires

$X174 single-stranded the activity

Aside from the process of DNA synthesiswhich

(192).

(DNA polymerase

teins

III,

elongation

factors

postulated primer

that

one of the "initiation"

(S), the role

We will

present

takes place in two stages. the DNA in such a way that to a double stranded than ten proteins

The first

requires

but more than just

(1,3,4),many Although

bacteriophage

synthesizes

#X174 replicative

stage,whichis

protein

it

is the

form synthesis

and ATP dependent,alters

and subsequently

This second stage reaction DNA polymerase

pro-

remains obscure.

the DNA may be re-isolated

form efficiently.

protein)

(dnaG protein)

proteins

ten proteins

four of these proteins

of DNA synthesis.

proteins

of the other remaining evidence that

of at least

and DNA binding

appear to be needed for the "initiation"

DNA to its

III

converted

requires

and elongation

less

factors.

MATERIALSAND METHODS Cells (Escherichia csH562) were grown in broth at 30°C, harvested, washed and frozen in a solution of 50mM Tris-HCl, pH7..5 containing 10% sucrose as previously described (6). Cell-free extracts were prepared as described by Wickner, Wright and Hurwitz (7). Routinely cell extracts contained 9-13 mg per ml protein. To render cell extracts stable and free of detectable deoxy or ribotriphosphates they were freed of nucleic acid and concentrated by ammonium sulfate precipitation. For each 1.0 ml of extract, 74 ul of neutralized saturated ammonium Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

506

Vol. 70, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

sulfate was added. The mixture was passed over a DEAE-cellulose column (equal in bed volume to the cell extract volume) which was previously equilibrated with S E D buffer (50 mM Tris-HCl,pH 7.5, 1 mM E D T A , 2 mM dithiothreitol and 10% sucrose) which also contained the same concentration of ammonium sulfate as the extract. The protein was collected (as judged by the yellow color) after passage through the column. The eluate was adjusted to 40% saturation with neutralized, saturated ammonium sulfate. After 10 minutes at O'C, the precipitate was collected by centrifugation and dissolved in the original volume of S E D buffer. The precipitation was repeated and the precipitate was washed with SED buffer containing ammonium sulfate (50% saturation), re-collected by centrifugation and finally dissolved in l/30 of the original extract volume of S E D buffer. This preparation (lo-12 mg/ml of protein) was used for the preparation of the "initiation complex" (see below) and also served as the starting extract for further purification of second-stage active components. Purification of "second-stage active" components was achieved by ammonium sulfate fractionation and phosphocellulose chromatography of the above fraction. Following precipitation of the activity at 40% saturated ammonium sulfate, the precipitate was extracted with S E D buffer (original fraction volume) containing ammonium sulfate at 35% saturation and then again with S E D at 25% saturation. The insoluble matter was finally dissolved in S E D but containing 20% glycerol and 20 mM NaCl in place of sucrose (l/5 original fraction volume). FolThis preparation contained 50-80% of the original second-stage activity. lowing dialysis, the active fraction was applied to a column of phosphocellulose equilibrated with the above buffer; 20-40% of the original activity is eluted with the same buffer containing 50 mu NaCl, Assays. For synthesis of 1$X174 RFII, the reactive mixture (50 ~1) was 20 mM Tris-HCl pH 7.5, 12 mM MgC12, 4 mM dithiothreitol, 2 mM in ATP and contained $X174 DNA, 100 p moles nucleotide; 2 n moles each dATP, dGTP and dCTP; 0.2 n moles and cell extract, 5 ul. of [3H] TTP (1.0 x lo3 cpm/p mole); 1.5 ug of rifampicin The incubation was at 30°C for varying amounts of time. Reactions were stopped by the addition of trichloroacetic acid (5%). Acid insoluble radioactivity was determined by liquid scintillation counting. Conditions for formation of the "initiation complex" were the same as above only all four deoxytriphosphates were omitted. Incubations were for 10 minutes at 3O'C. The complex was isolated by filtration through a column of Biogel A5M (0.5 x 10 cm) developed in buffer A (20 mM Tris-HCl pH 7.5, 1 mM E D T A , 12 mM MgC12, 4 mM dithiothreotol, 0.5 mM ATP and 2.5% sucrose). Complex was detected by adding 10 ul of deoxytriphosphates (same concentration as the assay conditions) to each fraction (50 1.11) and Incubations were for 20 minutes at 30°C. The reaction was 1 ul of original extract. stopped by the addition of trichloroacetic acid (5%) and 100 ug of bovine serum albumin was added as carrier. For preparation of large amounts of complex, the reaction volume and column dimensions were scaled up twenty-fold. Purified dnaG protein (880 units/ml, reference 7) was kindly provided by Dr. .Sue Wickner. Analysis of the product of the reactions were made by alkaline sucrose density gradient centrifugation as previously described (6). E. coli DNA polymerase III* and co-polymerase III* were purified phosphocellulose stage as described by Wickner et al, (8).

through

the

RESULTS The partially of converting

purified

single-stranded

extract,prepared bacteriophage

as described

in Methods,was capable

$X174 DNA to a double-stranded

507

(RFII)

Vol. 70, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1, z 8

- 20

-

P 15 * 0

-

IO

-

5

3-

& t g2-

I

F I- IF CL. 20 30 FRACTION

I I I IO 20 30 TIME (MIN.1

40 50 0 NUMBER

0

IO 20 30 40 FRACTION NUMBER

2. Figure 1. Bacteriophage $X174-dependent DNA synthesis. The components of the reaction mixture are given in the methods. Incubation was for 7 minutes at 30°C in the absence of dNTPs. Deoxytriphosphates are added at time zero (o--o--o). In the first incubation ATP (A--A--A) or DNA (O--O--Q was omitted and then added along with dNTPs in the second incubation. $X174 DNA synthesis in two stages. Following the Figure 2 . Bacteriophage first incubation (figure 1) reaction mixture was filtered through a Biogel ASM column (2A) . To each fraction was added dNTP and extract and incubation was as in figure 1. The arrows (left to right) represent the p,osition of elution of $X174 Marker DNA, hemogloblin and dNTP respectively. Panel B represents the kinetics of incorporation in the second stage. Panel C represents an alkaline sucrose density gradient analysis of the product of the second stage reaction. The arrow marks the position of circular fd DNA marker, The details are in the methods section.

form

in a rifampicin-resistant

reaction.

way from

dnaE and dnaG mutants

pared

extracts

to

Although

more

by 30 minutes is

a lag

of

or DNA is is that

It

still there

incubation,

except

addition

are

that

temperature-sensitive

input

DNA is

reaction

does

5 minutes the

lag

could

deoxytriphosphates

in

in the

first

prepared

in this

DNA synthesis

com-

place

the

first

the

reaction

and then

that

508

all

incubated proceeds added

to the

with

proceeds if

summarized lead

linearly

reaction

be abolished

The results, taking

to a double-stranded

proceed

were

incubation

incorporation.

two reactions

for

converted not

and then

of deoxytriphosphates,

omitted a lag

the

the for

extracts

dna+ cells.

80% of

was found

reaction

Following

than

from

in incorporation

25 minutes. of the

prepared

were

In addition,

after

five

linearly. five

in figure formation

time.

There

linearly

of the for

form

for

components minutes. If

minutes,there 1, indicate of double

ATP

Vol. 70, No. 2, 1976

stranded

BIOCHEMICAL

DNA. The first,

deoxytriphosphates.

slow

Following

AND BIOPHYSICAL

reaction

requires

proteins,

ATP and DNA but not

the first

reaction,

addition

of deoxytriphosphates

to the system promotes immediate and rapid These results

suggested that

ment for deoxytriphosphates. the first

incubation

gel filtration replicative

is a full

If

the

DNA is

for RF formation,

it

There is no lag in incorporation

length,

complementary

strand

re-isolated verting

essentially

formation

all

of the "treated

of only 2.5% of that

the activity concentration

of the extract

Thus it appears that than the overall

The amount of DNA synthesized

required

(l/10)

DNAintoa

is directly

This property

$@tivitycould

not be replaced

DNA (table

In addition

to convert

the

capable of conto catalyze

1).

the

Furthermore, dependence with

DNA shows a linear simpler

double-stranded

in its

dependence.

requirements

co-polymerase

III*

form requires

by the addition

proportional allowed

by the addition

all

of ribotriphosphates.

to the amount of extract

for the partial

(see methods).

purification

The "second stage"

of DNA polymerase III*

added were equal to that

amount present

and

in the

fraction was found to contain the dnaG protein *The "second stage" activity (> 42 units/ml, units defined in Ref. (7)) in addition to DNA polymerase III, E. coli unwinding protein and elongation factors. There was no detectable dnaB or dnaC(D) proteins (these assays were kindly performed by Dr. S. Wickner, National Institutes of Health).

509

and

for RF formation

is sufficient

is considerably

in the second stage*

(units

the requirements

on "treated"

and is not stimulated

added in the second step.

2C).

DNA shows an exponential

activity

reaction

is.

of the isolated

four deoxytriphosphates

(figure

I1 DNA to RF is sufficient

on untreated

tothe

of the reaction

The amount of extract

the second reaction

reaction

The conversion

the activity

rate,

amount on untreated

whereas the extract

the initial

The product

of the template

form.

by

promotes immediate DNA synthesis

a much reduced amount of extract DNA to a double stranded

the DNA following

subsequent conversion after

isolated

to the require-

and small molecules

for its

seen.

to the absence of a lag in the incorporation are such that

reaction,

from the bulk proteins

2A) and the requirements

form were studied.

28).

of DNA.

In order to examine this

then used as a substrate (figure

synthesis

the DNA had to be acted upon prior

was separated

(figure

RESEARCH COMMUNICATIONS

of

Vol. 70, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE It Activity

System Complex

+ extract

+ dNTP

loo%*

Complex

- extract

+ dNTP

0.1

Complex

+ extract

+ dATP, dGTP, -dCTP

6.7

Complex

+ extract

+ dNTP + rNTP

Complex

+ partially

- Complex

+ extract

purified

extract

(percent)

91.0 + dNTP

+ $X174 DNA + dNTP

65.0 2.5

-t In all two stage reactions the final concentrations of DNA, ATP and dNTPs are always the same as the standard incubation mixture (see methods). *

100% activity represents 7-10 p moles of 3H TTP incorporated. The amount of activity recovered in the two stage reaction was usually about 50% of the total activity possible for the given amount of DNA and extract.

TABLE II 1st Additions

2nd Additions

Activity

complete

complete

100%

complete - 10' at 30°C +lO'at 4OC

complete

32.0

complete - $X174 DNA + fd DNA

complete

co.1

complete - 4X174 DNA

complete + +X174 DNA

co.1

complete - ATP

complete +ATP

15.0

The first incubation (7 min. at 3O'C) contains all of the necessary components (see methods) for DNA synthesis except dNTPs. After gel filtration, dNTPs and extract are added and incubation is for 20 min. at 30°C. "First Additions" are to the first stage reaction. "Second Additions" are to the second stage reaction.

510

(percent)

BIOCHEMICAL

Vol. 70, No.. 2, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE III

System

Activity

Complex

+ dna+ extract

Complex

+ dnaGts extract*+

Complex

+ dnaGts extract

Complex

+ ~dnaGts extract +a18 units dnaG protein + dNTP at 37'C

* Prepared as described

extract

added) indicating

the synthesis

is that

of "active"

DNA to become active

of bacteriophage

"activation"(priming,

The second stage reaction As shown in Table III, in function

can be restored of purified

112

coli

stage reaction Further

of the treated

DNA in the first (Table II).

NY73.

did not just

evidence that

lead to a primer was

DNA to remain active

stage reaction

Furthermore

fd DNA in the first This result digestion,

decreased

etc.)

the activity

the ability

incubation

of the input

for $X174 DNA in that an "active"

the possibility

that

a non-

was occuring. requirement

of dnaGtS extract

when compared to extract

required

stage did not yield

makes unlikely

has an absolute

to the dnaGts extract

prepared

for the dnaG protein.

in the second stage is temperature from dna+ cells.

at the non-permissive

temperature

All activity by the addition

dnaG protein. --

The requirement formation

co.1

in the second stage was specific

DNA in the second reaction.

sensitive

the first

the ability

of the DNAwithATP and extract

specific

+ dNTP at 37'C

time at O'C of one hour.

The formation

substitution

34

in methods from Escherichia

that

100%

dNTP at 30°C

of a primer on the @X174 DNA.

not synthesized with a half

+ dNTP at 30°C or 37'C

(percent)

for the dnaG protein -

of "active"

from dnaGtS cells -

is seen only in the second stage,

DNA is not temperature

sensitive

(data not shown). 511

when prepared

in that

with extracts

Vol. 70, No. 2, 1976

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

DISCUSSION Our results

suggest that

replicative

form requires

association

of proteins

decays even at 0°C. (perhaps requiring length

before

three distinct

reactions.

strand.

this

hypothesis

protein

been shown that

Thus the first

"simply"

synthesize

first

It is not obviously

before

in subsequent

the *B,

stage is synthesis

(1).

its

of a full

components will

be

can be separated

This study showed that

-dnaC(D), X, Y, and Z proteins

and factors

and

generation)

apparent

an RNA primer as reported

priming

I and II were not required

appear to "prepare"

(perhaps for primer

stage even though it

are required

analysis

stage proteins

upon by the dnaG protein can act.

of a new DNA chain

the @X174 RF promotion

DNA polymerase III

proteins

and

had to be added to the $X174 DNA in the presence of ATP.

The dnaG protein, initially.

the

can be proven.

the lag for DNA synthesis,

DNA binding

involves

complex is labile

of the system into

into two stages on the basis of kinetic to abolish

this

and the third

Resolution

$X174 DNA to its

The first

with the DNA specifically;

the dnaG protein) -

It has already

of single-stranded

The second stage leads to initiation

complementary

required

the conversion

was present.

that

if

(S),why it

the DNA to be acted

and then the elongation the dnaG protein

had not acted in the

Perhaps the addition

can take place.

were to

These questions

of deoxynucleotides will

be addressed

studies. REFERENCES

1.

Wickner,

2.

Schekman, R., Weiner, J., 250,

S. and Hurwitz,

J. (1974).

Proc. Nat., Acad. Sci, USA 7l,

Weiner, A. and Kornberg,

A. (1975).

4120-4124.

J. Biol.

Chem,

5859-5865.

3.

Zechel,

K., Bouche, J.P. and Kornberg,

4.

Sherman, L.,

5.

Bouche, J.P.,

6.

Molineux, I.J., 6090-6098.

7.

Wickner, S., Wright, 70, 1613-1618.

8.

Wickner, W., Schekman. R. Geider, Acad. Sci. USA 70, 1764-1767.

Ph.D. thesis, Zechel,

Massachusetts

K: and Kornberg,

Friedman,

A. (1975). Institute A.

S. and Gefter,

M. and Hurwitz,

J,

(1975).

(1973).

Chem. 25Q, 4684-4689,

of Technology J, Biol.

M.L. (1974).

K. and Kornberg,

512

J, Biol.

(l976).

Chem. 250, 5995-6001,

J. Biol,

Chem. 249,

Proc. Nat. Acad., Sci. USA A, (1973).

Proc. Nat.