J. Mol. Biol. (1976) 100, 543-556

Bacteriophage T7 Deoxyribonucleic Acid Replication in Vitro Vt. Synthesis o f Intact C h r o m o s o m e s o f Bacteriophage T7 WARREN E. MASKER~ AND CHARLESC. I~IC~A~DSON Department of Biological Chemistry Harvard Medical School, Boston, Mass. 02115, U.S.A. (Received 4 July 1975, and in revised form 6 October 1975) Gently lysed extracts of Escherichia coli infected with bacteriophage T7 carry out extensive DNA synthesis in the presence of an exogenous T7 DNA template (Hinkle & Richardson, 1974). The amount of DNA synthesized in the in vitro reaction is two to three times the amount of template DNA added to the reaction. Analysis by zone sedimentation through alkaline sucrose gradients indicates that the products synthesized by extracts prepared from phage-infected E. coli mutants deficient in DNA polymerase I are considerably smaller than intact T7 DNA strands. However, when extracts of phage-infected wild-type E. coli are used in the in vitro reaction, the average molecular weight of the product DNA is increased; up to 50% of the DNA is the same length as intact T7 DNA. Such high molecular weight DNA is also synthesized when extracts of T7-infeeted E. coli polA1 mutants are supplemented with homogeneous E. coli DNA polymorase I. As judged by isopycnic analysis, the product DNA is not covalently attached to the template. The accompanying paper (Masker & Richardson, 1976) describes the biological activity of the high molecular weight DNA synthesized in the in vitro system.

1. Introduction Previous extensive genetic and biochemical characterizations of bacteriophage T7 (Studier, 1972) make this host-phage system especially suitable for studying DNA replication. Recently, cell-free systems have been developed t h a t support replication of duplex T7 D N A in vitro (Str~tling et al., 1973 ; Hinkle & Richardson, 1974). DNA synthesis in vitro requires the four deoxynucleoside triphosphates and an exogenous T7 D N A template; the rate of synthesis is increased several-fold by the four ribonucleoside triphosphates. Replication is dependent on several gene products necessary for D N A synthesis in rive, although independent of others. For instance, the products of phage genes 2, 3 and 6 are not required in vitro. The role of the product of gene 2 in D N A replication in rive has not yet been determined. Genes 3 and 6 specify nucleases t h a t are involved in the breakdown of host D N A and subsequent provision of nucleotide precursors for phage D N A synthesis (Center et al., 1970; Sadowski & Kerr, 1970; Sadowski, 1971). However, incorporation of deoxyribonucleotides into T7 D N A in vitro is dependent on the products of phage genes 4 and 5 as it is in rive. t Paper IV in this series is Hinkle & Richardson (1975). :~ Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 37830, U.S.A. 543

544

W . E . MASKER AN]) C. C. RICHARDSON

The specific role of the protein product of gene 4 is not k n o ~ l (Str~tling & Knippers, 1973; Hinkle & Richardson, 1975), but gene 5 is known to code for a subunit of T7 DNA polymerase (Grippo & Richardson, 1971 ; Oey et al., 1971 ; Moch'ich & Richardson 1975b). Like other in vitro replication systems, the T7 system has been particularly useful in devising complementation assays to purify and characterize phage- and hostspecified proteins important in the replication of T7 DNA. Using such a complementation assay the protein product of gene 4 has been purified (Strgtling & Knippers, 1973; Hinkle & Richardson, 1975) and shown to stimulate DNA synthesis by the T7 DNA polymerase when native T7 DNA is used as template (Hinkle & Richardson, 1975). Similarly, the in vitro system was helpful in identifying and purifying (Modrich & Richardson, 1975a) a host protein, the product of the lsnC gene of Escherichia coli (Chamberlin, 1974) essential for T7 DNA replication. The purified TsnC protein is a subunit of the T7 DNA polymerase (Modrich & Richardson, 1975b) and is identical to E. coli thioredoxin (Mark & Richardson, unpublished results). DNA synthesis in the in vitro systems is linear over a 20 to 30-minute period and occurs by a semi-conservative mechanism (Strgtling et al., 1973 ; Hinkle & Richardson, 1974). Under optimal conditions the amount of DNA synthesized is two to three times the amount of template DNA added to the reaction, and the product is not covalently linked to the template (Hinkle & Richardson, 1974). However, the DNA synthesizing in vitro consists mostly of small 11 S fragments corresponding in size to the intermediates of discontinuous synthesis observed during the synthesis of the T7 chromosome (Masamune et al., 1971). Possibly a component necessary for the joining of these "Okazaki fragments" (Okazaki et al., 1968) is lacking in the in vitro system. Early studies of in vitro T7 DNA replication sought to avoid repair-like DNA synthesis by using only extracts of phage-infected bacteria deficient in both endonuclease I and DNA polymerase I (Str~ttling et al., 1973 ; HIi~lkle & Richardson, 1974). However, previous studies of DNA replication in vivo had shown that "Okazaki fragments" are joined more slowly in E. coli polA mutants (deficient in DNA polymerase I) than in wild-type cells (Kuempel & Veomett, 1970; Okazaki el al., 1971). Thus, the accumulation of Okazaki fragments observed in vitro m a y be due to reduced levels of DNA polymerase I. Alternatively, DNA polymerase I might be needed in vitro to repair damage resulting fl'om nuclease activity in the extracts (Kanner & Hanawalt, 1970; Monk et al., 1971). In this paper we show that up to 50% of the T7 DNA synthesized i n vitro in the presence of DNA polymerase I has the same molecular weight as an intact T7 chromosome. In the accompanying paper (Masker & Richardson, 1976) we show that the high molecular weight DNA synthesized in vitro is biologically active and that it can transfect E. coli spheroplasts to form T7 phage. 2. Materials and M e t h o d s (a) Bacterial strains E. eoli D l l 0 s u - thy polA1 end, E. cell W3110 s u - thy, and E. cell 011' su + t h y h a v e been previously described (Moses & Richardson, 1970; DeLucia & Cairns, 1969; Studier, 1969). E. coli DR110 su - thy polA + end is a spontaneous revertant of E. cell D110 selected f01' ability to grow on L-broth agar plates supplemented with 0.04% (v/v) methylmethanesulfonate. The revertant was lacking suppressor I and III activity as determined by its

PHAGE

T7 DNA R E P L I C A T I O N

117 V I T R O :

V

545

ability to support the growth of amber m u t a n t s of bacteriophage lambda. E x t r a c t s of

E. coli D R l l 0 prepared by sonic irradiation were assayed for D N A polymerase I and found to contain 80~/o of the activity fotmd in similar extracts of E. coli W3110. (b) Bacteriophages T7 amber n m t a u ts were obtained from Dr F. W. Studier and were grown on E. coli 011' as described by Studier (1969). The amber mutations used in this study are gone 3, am29; gene 5, am28; and gene 6, aml47. I n the text, T7 m u t a n t s arc designated by subscript notation indicating gene only. For example, a double m u t an t , am29 and am147, is designated as TT.:.~, (c) Media L-broth, T-broth, and M9 media are described by Miller (1972), L-broth and T-broth were routinely supplemented with 0.1% glucose and 10 tLg thymine/ml. Fo r radioactive labeling of D N A in thymine-requiring strains, M9 minimal m e d i u m was supplemented with 2 tLg t h y m i n e / m l and 1 t~Ci [3H]thymidine/ml. For density labeling, 99.3 atomic ~o lSNH4CI replaced 14NI-I4C1; and 1 g 50~o enriched [13C]glucose/1 was used as a carbon source in M9 medium. Tryptone, yeast extract, and glucose were purchased from Difco. (d) Other materials Unlabeled nucleotides were ptu'chased from Schwarz-Mann Bioresearch. [a-32P]dATP and [aH]dTTP were obtained from New England Nuclear. [methyl-3H]thymidine was obtained from New E n g l a n d Nuclear. ~SNH4C1 was obtained from BioRad Laboratories. [laC]glueose was purchased from Merck, Sharp and Dohme, Lt d of Canada. Lysozyme was purchased from Worthington. Ultrapure sucrose was from Schwarz/Mann. D N A polymerase I was purified as described by J o v i n et al. (1969) ; the Sephadex G100 fraction had a spee. act. of 17,500 units per mg protein.

(e) Preparation of T7-infected bacteria T7 phage-infected E. coli were prepared as described previously (Hinkle & Richardson, 1974} except for the following modifications: phage infection was for 14.5 rain at 37~ After eentrifugation the phage-infected cells were resuspended in 50 mM-Tris-HC1 (pH 7"5), 10% sucrose at a conen of 9.5 • 10 l~ cells/ml, and 2.1 ml portions were frozen and stored in liquid nitrogen. (f) Preparation of cell extract Cell extracts were prepared by a modification of the m e t h o d of Wickner et al. (1972). Frozen cell preparations (2.1 ml) were thawed at room temperature, and 0.042 ml 5 ~NaC1, 0-042 ml of lysozyme (10 mg/ml) in 50 m~-Tris.HC1 (pH 7.5) and 10~ sucrose were added. After incubation at 0~ for 45 min, and at 37~ for 2 min, the lysate was centrifuged at 4~ in the Spinco 40 rotor at 39,000 r p m as described by Hinkle & Richardson (1974). The extracts were routinely adjusted to an A280 of 85 with 50 mM-Tris-HC1, 0-1 M-NaC1, I0~o sucrose. All experiments were performed with freshly prepared extracts. (g) Assay for D N A synthesis Replication of duplex T7 D N A in vitro was assayed according to Hinkle & Richardson (1974) except t h a t the final concentration of Tris.HC1 (pH 7.5) in the reaction mixture was 30 mM, and 3 nmols of T7 D N A were added to the 0.1 ml reaction mixture. (h) Preparation of D N A Unlabeled and 3H-labeled T7 D N A were prepared from wild-type T7 phage grown on strain 011' as described by Richardson (1966). To prepare h e a v y density DNA, E. coli 011' was g r o ~ l in M9 medium which contained 15NH4C1, [13C]glueose and [all]thymine. In this m e d i u m optimal recovery of phage was achieved by infecting the bacteria witll

546

W.E.

MASKER

AND

C. C. R I C H A R D S O N

wild-type T7 at a multiplicity of infection of 0-1 at an As~0 of 0.6. Under these conditions titres of 2 to 3 • 101~ plaque-forn~ng units per ml were obtained. The D N A prepared from these phage had a density of 1.745 g / c a 3 as estimated from equilibrium sedimentation in CsC1 gradients. All concentrations of DNA are expressed as equivalents of nucleotide phosphorus. (i) Zone sedimentation in neutral or alkaline sucrose Sucrose gradients were prepared from tfltrapure sucrose as described by Hinkle & Richardson (1974). The celhilose nitrate tubes were routinely washed with a solution of 1 n ~ - E D T A - 1 0 / ~ g denatured salmon sperm DNA/ml, followed b y extensive washing with distilled water. Prior to centrifugation, reaction mixttLres of 0.1 ml were incubated at 42~ for 1 0 m i n with l0 ~1 of 0-5 ~a-EDTA a n d 0.1 ml of 20mM-Tris.HC1 (pH 7.5) -1 M-l~aC1-4~ Sarkosyl NL97. Samples (0-1 ml) were layered onto 5 ml sucrose gradients and centrifuged at 49,000 r p m in the Spinco SW50.1 rotor at 4~ for 150 min. For preparative runs, up to 1-0 ml was layered on a 38 ml gradient a n d centrifuged at 24,000 r p m in the Spinco SW27 rotor at 4~ for 15 h. I n all Figures the direction of sedimentation is from right to left. Unless otherwise stated, the data are plotted so t h a t a u n i t distance on the ordinate corresponds to the same n u m b e r of picomoles of 3H- or 32P-labeled DNA. (j) Isopycnic eentrifugation Equilibrium sedimentation in neutral or alkaline CsC1 was performed by a modification of the method described by H a n a w a l t & Cooper (1971). DNA synthesis in a 0-2 ml reaction mixture was terminated by chilling the reaction mixture in ice and adding 1.8 ml of 10 m ~ -Tris.HC1 (pH 8-0), 5 mM-EDTA. For neutral CsC1 gradients, 1 ml of this mixture a n d 5 m l of 10mM-Tris.HCl, 1 m ~ - E D T A , 10m~-NaC1 were added to 8 g CsC1. For the alkaline gradients, 0-7 ml of potassium phosphate buffer (pH 12-0) was added to the remaining 1.0 ml of diluted reaction mixture to which 8-0 g of CsC1 and 4-3 ml of 10 rn~Tris.HC1, 1 mM-EDTA, 10 mM-NaC1 were then added. The gradients were centrifuged at 33,000 rpm in the Spinco 40 rotor at 25~ for 60 to 65 h. Fractions of 0.13 ml were collected through the bottom of the centrifuge tube. (k) Determination of D N A content of gradients Before acid-precipitation, 50 ~1 of salmon sperm DNA (0-5 mg/ml) were added to fractions collected from sucrose or CsC1 gradients. The DNA in the fractions was precipitated b y the addition of 4 ml of ice-cold 1 ~-HC1, 0.1 M-sodium pyrophosphate. The precipitates were collected on W h a t m a n GF/C filters, and the radioactivity was determined as described by ttinkle & Richardson (1974). (1) Other methods Estimates of the a m o u n t of Dl~A polymerase I present in crude extracts were obtained by modification of the assay described b y Richardson etal. (1964) using activated calf t h y m u s DNA or denatured salmon sperm DNA as a template. W h e n necessary,/V-ethylmaleimide (1-5 mM) was added to inhibit T7 DNA polymerase. In vivo pulse labeling of I)NA from T7-infected E. eoli and lysis b y alkali of pulse-labeled cells were performed as described by Masamune etal. (1971). Fraction I I of the in vitro replication system was prepared by streptomycin sulfate a n d a m m o n i u m sulfate precipitation of extracts as described by Hinkle & Richardson (1974).

3. Results (a) Kinetics of synthesis and size distribution of D1VA synthesized by extracts of pha4]e-infected polA + a n d p o l A - hosts The kinetics of T7 D N A synthesis i n in vitro r e p l i c a t i o n s y s t e m s are similar with b o t h T7-infeeted poIA + a n d p o l A - strains as s h o ~ l i n F i g u r e 1. E. coli D l l 0 poIA1,

PHAGE

T7 DNA

REPLICATION

117 V I T R O :

V

547

.c

"/A//2"

w

-~- . . . . . . . . . . . . . . . . .

/

9--

/

0

//

I I0

1 20

I

30 Time

~ 40

I

50

(min)

FzO. 1. T h e k i n e t i c s o f in vitro T7 D N A s y n t h e s i s . E x t r a c t s o f T73.6-1nfected E . coli W 3 1 1 0 , D R l l 0 , a n d D l l 0 were p r e p a r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e k i n e t i c s o f D N A s y n t h e s i s were m e a s u r e d u s i n g t h e s t a n d a r d a s s a y c o n d i t i o n s d e s c r i b e d in M a t e r i a l s a n d M e t h o d s a n d in t h e l e g e n d to Fig. 2. [ a - 3 2 P ] d A T P w a s p r e s e n t a t 8.0 e p m / p m o l . - - O O - - , T7~.6i n f e c t e d E. coli W 3 1 1 0 ; - - [ 2 ] - - [2]--, T73,6-infected E . coli D R l l 0 ; - - A - - A - - , T73.6-infected E. coli D l l 0 .

deficient in DNA polymerase I, is compared to E. coli D R l l 0 (a p o l A + revertant of E. coli Dll0) and to E. coli W3110, a wild-type strain. Although the kinetics of synthesis is similar in the p o l A + and p o l A - strains, the initial rate of synthesis and the extent of synthesis are 1.5 to 2-fold higher in extracts of cells that contain normal levels of DNA polymerase I. However, a striking effect of the presence of polymerase I in the replication system was observed when the size of the product was determined by zone sedimentation through alkaline sucrose density gradients. In agreement with our earlier results (Hinkle & Richardson, 1974), DNA synthesized over a 20-minute period in extracts of T73,6-infected E. coli Dll0, lacking polymerase I, consists mostly of pieces considerably smaller than intact T7 DNA molecules (Fig. 2(a)); only approximately 13% of the product DNA (fractions 11 to 16) sediments with the phage-size T7 DNA. In contrast, a large percentage (57%, fractions 11 to 16) of the DNA synthesized in an extract prepared from T73.8-infeeted E. coli DRll0, a revertant containing polymerase I, sedimented in alkali near the position expected for intact T7 DNA strands (Fig. 2(b)). From these two profiles it also appears that the template strands contain fewer breaks when DNA polymerase I is present during the incubation. Since the polymerase I in E. coli D R l l 0 is probably not fully wild-type, a more detailed analysis of the DNA synthesized in the presence of polymerase I has been carried out using extracts of T7-infected E . coli W3110 p o l A + . After 3, 10, 20 and 40 minutes of incubation, samples were removed from the reaction mixture, and the DNA was analyzed by zone sedimentation through alkaline sucrose gradients (Fig. 3). The DNA synthesized at 3 minutes is mostly of low molecular weight. After 10 minutes and 20 minutes, the average molecular weight of the product has increased, and some newly synthesized material sediments at the same position as the 3H-labeled template. By 40 minutes the amount of high molecular weight DNA exceeds the

548

W. E. MASKER

AND

C. C. R I C H A R D S O N

(o)

20

6

i(k~,

4 _

i

10 2

r

0

0 I0

x

E u

(b)

ff

{L {.

t~

~

e

,, :

20 ,

4

2

o I0

20

Freclion

30

o

rlo~

Fro. 2. Size distribution in alkali of T7 DNA synthesized in vitro by extracts of T7z/;-infected polA + and polA- strains. Extracts of T7ja-infected E. coli D l l 0 polA- (a) and T7z.,;-infected E. coli DRI10 polA + (b) were prepared as described in Materials and Methods. 10 /~1 of each extract of T7.~.a-infected E. cell was added to separate reaction mixtures (0'05 ml final vol.) containing 30 mM-Tris.HCl (pH 7.5), 20 m~-MgC12, 10 mM-2-mercaptoethanol, 0-3 mM of each of the four rNTPs, 0-3 mM each of dCTP, dTTP, dGTP, [u-a2pJdATP (44.8 cpm/pmol), and 1-5 nmol of wild-type T7[OH]DNA (3-5 cpm]pmol). After 20 min of incubation at 30~ the reactions were stopped by adding 10 /~l of 0.5 M-EDTA. After incubation with Tris.HCl, NaC1, Sarkosyl as described in Materials and Methods, 0-1 ml sampIes were layered on alkaline sucrose gradients as described. The results shown were obtained with T7:~.a-infected E. coli DI10 (a) and E. coli D R l l 0 (b). - - A - - A - - , 3H-labeled template DNA; - - 0 O - - , a2P-laboled product DNA (11.2 cpm/pmol DNA). a m o u n t of t e m p l a t e D N A . T h u s e x t r a c t s o f T7~,e-infected W 3 1 1 0 c a n c a r r y o u t a n e t s y n t h e s i s o f p h a g e size T7. (b) Comple~entation of extracts of phage-infected p o l A - cells with purified D N A polymerase I W e h a v e e x a m i n e d t h e effects of a d d i n g purified D N A p o l y m e r a s e I to e x t r a c t s o f p h a g e - i n f e c t e d poIA cells in o r d e r m o r e clearly to a t t r i b u t e o u r results to t h e presence o f this enzyme. W h e n h o m o g e n e o u s D N A p o l y m e r a s e (1 unit/0.05 m l o f r e a c t i o n m i x t u r e ) was used to c o m p l e m e n t t h e r e p l i c a t i o n s y s t e m p r e p a r e d from a n e x t r a c t of E. coli D l l 0 p o I A - - i n f e c t e d cells, t h e size d i s t r i b u t i o n o f t h e p r o d u c t was similar t o t h a t p r o d u c e d b y e x t r a c t s of p h a g e - i n f e c t e d E. coli polA + cells (Fig. 4). Again, in t h e presence of D N A p o l y m e r a s e I t h e t e m p l a t e s t r a n d s h a v e fewer breaks. T h e a m o u n t o f D N A p o l y m e r a s e I a d d e d in this e x p e r i m e n t is a p p r o x i m a t e l y t h a t n o r m a l l y f o u n d in e x t r a c t s o f E. coli D R l l 0 p r e p a r e d in a n i d e n t i c a l m a n n e r . These results i n d i c a t e t h a t t h e i n c r e a s e d size o f t h e n e w l y s y n t h e s i z e d D N A in e x t r a c t s o f E. coli poIA +-infected cells results from t h e presence o f D N A p o l y m e r a s e I in t h e in vitro r e p l i c a t i o n s y s t e m .

P H A G E T7 D N A R E P L I C A T I O N 3 rain

15

~'~ ~ mt J

549

! 0 rain

8

4

5

"~

V

lO

,o X

IN VITRO:

2

w

o x E u

o x

gO rain

E o. u

12

v

z

15

15

"~

10 8 10

10

6 5

4 2

.~,~10

20

30

0

~

0 10

,

, -4=~ ...... *kL

20

30

Fraction no.

FIC. 3. Size distribution in alkali of T7 DNA synthesized in vitro by an extract of T7a.6-infeeted E. coli W3110. The extract was prepared as described in Materials and Methods, and 40 ~l were incubated in a 0-2-ml standard reaction mixture as described in the legend to Fig. 2. After the indicated times of incubation at 30~ 0.05-ml samples were withdrawn and analyzed by sedimentation through alkaline sucrose gradients as described in the legend to :Fig. 2 and Materials and Methods. - - O O - - , 32P-labeled product DNA (4-4 cpm/pmo[ DNA); - - ~ - - ~ - - , alllabeled template DNA (3-1 epm]pmol DNA).

(c) High molecular weight D N A arises by semi-conservative replication of exogenous D N A W e h a v e c a r r i e d o u t a n u m b e r of e x p e r i m e n t s t o show t h a t t h e phage-size T7 D N A s y n t h e s i z e d in t h e presence o f D N A p o l y m e r a s e I is t h e r e s u l t of semi-cons e r v a t i v e r e p l i c a t i o n of exogenous T7 D N A t e m p l a t e , a n d does n o t arise s i m p l y from r e p a i r o f c o n t a m i n a t i n g r e p l i c a t i n g D N A i n t e r m e d i a t e s p r e s e n t in t h e cells w h e n e x t r a c t s were p r e p a r e d . F i r s t , we h a v e d e t e r m i n e d t h e a m o u n t o f D N A s y n t h e s i s in t h e presence a n d a b s e n c e of exogenous T7 D N A t e m p l a t e b o t h in t h e presence a n d absence of D N A p o l y m e r a s e I. T h e results, s h o w n in T a b l e ], i n d i c a t e t h a t e x t r a c t s c o n t a i n i n g D N A p o l y m e r a s e I show less d e p e n d e n c e u p o n exogenous t e m p l a t e , b u t t h a t t h e a d d i t i o n o f exogenous D N A increases s y n t h e s i s to a p p r o x i m a t e l y t h e s a m e e x t e n t in all cases. T h e i n c r e a s e d a m o u n t of D N A s y n t h e s i s in t h e presence or a b s e n c e o f exogenous D N A in e x t r a c t s p r e p a r e d f r o m E. coli W3110 p o l A + r e l a t i v e to t h e o t h e r e x t r a c t s listed in T a b l e 1 m a y reflect a r e p a i r - t y p e s y n t h e s i s c a t a l y z e d b y p o l y m e r a s e I in which n u c l e o t i d e s are a d d e d t o t h e 3 ' - t e r m i n i of f r a g m e n t e d endogenous D N A . T h e lower a m o u n t s of s y n t h e s i s o b s e r v e d in t h e r e v e r t a n t D R l l 0 polA + could be due to t h e lower specific a c t i v i t y of this m u t a n t enzyme. S i m i l a r l y t h e a d d i t i o n of p o l y m e r a s e I to t h e polA - e x t r a c t m a y n o t h a v e r e s t o r e d t h e n o r m a l level o f t h e e n z y m e f o u n d in t h e p o l A + e x t r a c t . I n o r d e r to rule o u t specifically t h a t t h e high m o l e c u l a r w e i g h t D N A s y n t h e s i z e d

550

W. E. MASKER

A N D C. C. R I C H A R D S O N

(o) I0

c~ I

E

0 x

--

(b)

u

2

A 10

I0

20

30

Froc lion no

F i e . 4. Alkaline sucrose sedimentation of T7 DNA synthesized in vitro in the presence (a) and absence (b) of DNA polymerase I. An extract of T7:~,,;-infected E. cell D l l 0 was used in identical 0.05-ml assay mixtures except t h a t one mixture contained one unit of DNA polymeraso I (1 unit] 0-05-ml reaction mixture). After 20 min incubation at 30~ the mixtures were sedimented through alkaline sucrose gradients as described in the legend to Fig. 2. The profile of radioactivity from the gradient with the reaction mixture t h a t did not contain additional DNA polymerase I is shown in (a). - - / ~ - - A - - , 3H-labeled template DNA (2-8 cpm]pmol DNA); - - O Q - - , a2p-labeled product DNA (5"0 cpm]pmol DNA).

TABLE 1 Dependence of i n v i t r o D N A E. coli strain

synthesis on exogenous template

D N A synthesis -- Exogenous -{- Exogenous template template

-t- Template] -- Template

(nmol/20 min)

(nmol/20 min)

W3110 polA +

3"1

6.5

2.1

D R l l 0 polA +

1-3

4-9

3-8

D l l 0 polA- + polymerase I

0.84

3-8

4-5

Dll0 polA-

0.56

3.9

7.0

E x t r a c t s of T7~.a-infeeted E. coli W3110, D R l l 0 , and D l l 0 were prepared as described in Materials and Methods. The extracts were incubated in O-l-ml s t a n d a r d reaction mixtures in the presence or absence of 3 nmol of T7 DNA. The extract of T7a,a-infeeted E. coli D 110 was incubated in the presence or absence of 2 units of DNA polymerase I per reaction mixture. All reactions contained [~-32P]dATP (8 epm]pmol). After 20 min at 30~ the reactions were stopped, the DNA was precipitated, and the radioactivity was determined as described in Materials and Methods.

P H A G E T7 D N A R E P L I C A T I O N

IN VITRO: V

G51

in vitro results from replication of endogenous D N A , we examined the size distribution

of D N A synthesized in the absence of exogenous template. The reaction was carried o u t using an e x t r a c t of E. coli D l l 0 infected with T7s.~ with the addition of purified D N A polymerase in the presence and absence of 3 nmol of T 7 [ 3 H ] D N A template. After 20 minutes o f incubation the D N A was a n a l y z e d b y alkaline sucrose sedimentation. As shown in Figure 5, the small a m o u n t of D N A synthesized in the absence of exogenous t e m p l a t e has a significantly lower molecular weight t h a n the T 7 [ 3 H ] D N A a d d e d as a marker. Therefore, replication of endogenous D N A does n o t contribute significantly to the high molecular weight p r o d u c t synthesized in the presence of D N A polymerase I. Second, u n d e r conditions where the in vitro replication system is unable to replicate T7 D N A , only limited synthesis occurs in the presence o f D N A polymerase I, and no high molecular weight D N A is synthesized. E x t r a c t s of T7s,5,~-infeeted E. cell D l l 0 c a n n o t replicate T7 D N A as a result of the deficiency in T7 D N A polymerase. W h e n such an e x t r a c t was c o m p l e m e n t e d ~dth E. coli D N A polymerase I, only

(o)

I0

Template 8

zh

Product

6 4 2

2

-

qx

0

~

E

a.

x

(b)

20

4 ~

T7 Marker DNA

I

15

!

,

!

10

E

\

!

!

I

tO

N~,

20 Fraction

Product

30

no.

FIe. 5. Alkaline sucrose sedimentation of DNA synthesized in vitro in the presence or absence of exogenous template. An extract of T7z,rinfected E. cell Dll0 was incubated in a standard reaction mixtt~re with (a) and without (b) exogenous [3H]DNA template (see text). After the samples were layered on alkaline sucrose gradients, T7[3H]DNA was added as a marker to the gradient without exogenous template (b). The profile of the gradient with exogenous template is shown in (a). In order to emphasize the size distribution of the DNA synthesized from endogenous template, the convention of having equal vertical distances represent equal numbers of pmoles was not followed in (b). - - A - - A - - , 3H-labeled DNA (3.1 cpm/pmol DNA); --0 O - - , 32P-labeled DNA (2.3 cpm/pmol DNA).

552

W.E.

M A S K E R A N D C. C. R I C H A R D S O N

l i m i t e d D N A s y n t h e s i s o c c u r r e d (less t h a n 10% of t h a t o b s e r v e d in T7z,6-infected cell e x t r a c t s ) , whereas t h e a d d i t i o n o f purified T7 D N A p o l y m e r a s e r e s u l t e d in full r e s t o r a t i o n o f D N A synthesis. F u r t h e r m o r e , n o n e o f t h e r a d i o a c t i v e p r o d u c t sedim e n t e d f a s t e r t h a n 11 S i n alkaline sucrose g r a d i e n t s ( d a t a n o t shown). F i n a l l y , to c h a r a c t e r i z e f u r t h e r t h e D N A s y n t h e s i z e d in t h e presence o f D N A p o l y m e r a s e I, d e n s i t y - l a b e l e d T7[3H,~SN,~3C]DNA has been used as t e m p l a t e , a n d t h e p r o d u c t has been a n a l y z e d b y s e d i m e n t a t i o n to e q u i l i b r i u m in a CsC1 d e n s i t y g r a d i e n t . R e a c t i o n s were carried o u t for 20 m i n u t e s in t h e presence of e x t r a c t s p r e p a r e d f r o m T7z.e-infected E . coli D l l 0 s u p p l e m e n t e d w i t h D N A p o l y m e r a s e I a n d [~-32P]dATP. P y c n o g r a p h i c a n a l y s e s were p e r f o r m e d b o t h in n e u t r a l a n d a l k a l i n e CsCI (Fig. 6). U n d e r n o n - d e n a t u r i n g c o n d i t i o n s t h e m a j o r i t y of t h e 32P-labeled p r o d u c t b a n d s a t t h e d e n s i t y o f fully light d u p l e x D N A , a n d a significant p o r t i o n b a n d s t o g e t h e r w i t h a n equal a m o u n t o f 3H-labeled t e m p l a t e a t t h e p o s i t i o n o f h e a v y - l i g h t h y b r i d

25

(o)

20

15

I0

5

/ A t~

20

40

50

60

50

0

--

q 0

a.

(b)

25

E

ZO 15

o

i\

'~

f\

5

IO

30

20 Fraction

40

no.

FIe. 6. Isopyenic analysis of T7 DNA synthesized in vitro. An extract of E. coli D l l 0 infected with T%,~ was incubated with 4 units of DNA polymerase I in a 0.2-ml standard reaction mixtm'e as described in the legend to Fig. 2. Incubation was at 30~ for 25 min. The reaction was stopped and CsC1 gradients were formed as described in Materials and Methods. A total of 75 fractions were collected. The profile of acid-insoluble radioactivity in 50-/A samples of fractions recovered from the neutral gradient is shown in (a). Measurements of refractive indices on a similar gradient indicate that heavy DNA (1.745 g/era a) sediments near fraction 25; hybrid DNA is near fraction 35; and light DNA (1.710 g]cm s) is near fraction 46. A total of 58 fractions was collected from the alkaline gradient, and acid-precipitated to yield the profile in (b). - - / ~ - - • - - , aH,15N,laC-labeled template DNA (2.9 epm]pmol); - - O O - - , 32P-IabeIed product DNA (11 cpm/pmol).

P H A G E T7 D N A R E P L I C A T I O N

IN

VITRO:

V

553

D N A molecules. N o significant a m o u n t of a2P-labeled p r o d u c t is p r e s e n t in t h i s h e a v y region of t h e g r a d i e n t . P y c n o g r a p h i c a n a l y s i s o f t h e D N A in a l k a l i n e CsC1 r e v e a l s t h a t n o n e o f t h e p r o d u c t is c o v a l e n t l y l i n k e d t o large pieces o f t h e t e m p l a t e D N A . All o f t h e a2P-labeled p r o d u c t b a n d s a t t h e p o s i t i o n e x p e c t e d for light single s t r a n d s o f D N A , w h e r e a s all of t h e a l l - l a b e l e d t e m p l a t e b a n d s a t t h e p o s i t i o n e x p e c t e d for p S N , l a C ] D N A s t r a n d s . I n p a r a l l e l control e x p e r i m e n t s (not shown) in which D N A p o l y m e r a s e I was o m i t t e d from t h e r e a c t i o n m i x t u r e , i d e n t i c a l p y c n o g r a p h i c results were o b t a i n e d , in agreem e n t w i t h our earlier results ( H i n k l e & R i c h a r d s o n , 1974). S a m p l e s of t h e r e a c t i o n s were also a n a l y z e d b y zone s e d i m e n t a t i o n in a l k a l i n e sucrose g r a d i e n t s . T h e profiles o f t h e s e g r a d i e n t s (not shown) confirm t h e r e s u l t s h o w n in F i g u r e 4 ; t h e presence of D N A p o l y m e r a s e I increases t h e a v e r a g e m o l e c u l a r weight o f t h e p r o d u c t D N A to y i e l d phage-size D N A s t r a n d s . To show conclusively t h a t t h e high m o l e c u l a r w e i g h t D N A does n o t arise f r o m r e p a i r synthesis, t h e phage-size D N A s y n t h e s i z e d i n vitro w i t h T 7 [ a H , l a C , l S N ] D N A as t e m p l a t e h a s b e e n i s o l a t e d f r o m a n a l k a l i n e sucrose g r a d i e n t a n d a n a l y z e d in a l k a l i n e CsC1. As s h o w n in F i g u r e 7, t h e light p r o d u c t a n d h e a v y t e m p l a t e were c o m p l e t e l y s e p a r a t e d . W e conclude t h a t t h e high m o l e c u l a r w e i g h t D N A arises t h r o u g h a m e c h a n i s m o f s e m i - c o n s e r v a t i v e replication. F u r t h e r m o r e , r e p a i r of exogenous or

Alkoline s u c r o s e

N

'_o

6

0

~

,,

I~

l

-

~

-

~

o

1 ,a .-=_

Io

g n-

z

,s

zo

as

o

#

Fract{ . . . . .

~_

/J

~t

%.

8

~

:~

A

0

10

20

30

40

50

/

60

F r a c t i o n no.

Fro. 7. Isopycnic analysis of high molecular weight T7 DNA synthesized in vitro. A n extract of T7a.~-infected E. coli D110 was prepared as described in Materials and Methods and incubated at 30~ for 20 rain in a 0.6-ml standard reaction mixture which included 12 units of DNA polymerase I and T7[aH,lSN,laC]DNA as template. The reaction was stopped by the addition of 0.06 ml of 0.5 M-EDTA, and then 0'3 ml of 40 mM-Tris.HC1 (pH 7.5), 2 M-NaCI, 8% Sarkosyl NL97 was added. After incubation for 10 min at 42~ the entire sample was layered on a 36-ml 5% to 20% alkaline sucrose gradient formed over a 2-ml 70% sucrose shelf. The gradient was centrifuged at 4~ in the Spinco SW27 rotor for 15 h. A total of 66 fractions were collected, and a 75-1zlsample of each fraction was acid-precipitated to give the profile shown. The remainder of fractions 23 to 29 inclusive were pooled and dialyzed against three 2-1 changes of 50 raM-potassium phosphate buffer (pH 12.0), 10 mM-NaCl, 1 mM-EDTA. A CsC1 gradient was formed by adding the dialyzed fractions plus 0.4 ml I M-potassium phosphate (pH 12.0) plus sufficient buffer to total 6.0 ml per 8.0 g of CsC1. After equilibrium sedimentation the gradient was collected to yield 40 fractions, and 20-~1 samples of the fractions were acid-precipitated to give the profile of radioactivity shown in the insert. - - A - - A - - , aH,lSN,laC-labeled template DNA (3.4 epm/pmol D N A ) ; - - 0 O--, a2P-labeled product DNA (3.8 cpm/pmol DNA).

55 #.

W. E. MASKER

AND

C. C. R I C H A R D S O N

endogenous DNA templates does not contribute measurably to this species of DNA. (d) Size distribution of T7 D N A synthesized in vivo In contrast to the results obtained in the in vitro replication system, the deficiency in polymerase I in E. coli poIA1 has no detectable effect on the size of T7 DNA synthesized in vivo at various times. The procedure described by Masamune et al. (1971) was used to grow and label cultures of E. coli Dll0, DRll0, and W3110 infected with T73,6 or wild-type T7. Completion of high molecular weight DNA (of the size expected for an intact T7 clu'omosome) was slower in cells infected with T7~,6 than ceils infected with wild-type T7. However, these in vivo results (not shown) indicate no appreciable difference between polA § and p o l A - bacteria in the time required to synthesize phage-size T7 DNA after infection by ~dld-type T7 or T7j, 6 phage. 4. Discussion The in vitro replication system for T7 DNA synthesis resembles in vivo replication in several respects. However, a disturbing feature was the observation that the newly synthesized DNA was of low molecular weight (Str~tling et al., 1973; Hinkle & Richardson, 1974). The initial rationale for using extracts lacking polymerase I was to eliminate any non-specific repair-type activity which might mask T7 DNA replication. Further, T7 DNA replication in vivo did not appear altered in polA1 hosts. Our present results demonstrate, however, that DNA polymerase I is required for the in vitro synthesis of intact full-size T7 DNA molecules. A major concern in the present studies was that the radioactivity incorporated into the high molecular weight product in the presence of DNA polymerase I might represent a repair-type reaction with exogenous or endogenous T7 DNA template. However, several lines of evidence suggest that this is not the case. Pyenographic analyses using density-labeled template failed to show any covalent association of product with exogenous template. In addition the extensive synthesis observed and the relatively low molecular weight of the DNA synthesized in the absence of exogenous template suggest that endogenous DNA does not contribute significantly to the high molecular weight DNA s)mthesized in vitro. Hinkle & Richardson (1974) were able to prepare an ammonium sulfate fraction (fraction II), which was devoid of endogenous DNA and carried out T7 DNA replication in vitro. However, we find that fraction II will not synthesize high molecular weight DNA even in the presence of polymerase I. Dialysis of the extracts, an essential step in the preparation of fraction II, appears to limit their ability to synthesize high molecular weight DNA (Masker & Richardson, unpublished results). It is not clear how DNA polymerase I acts to increase the average molecular weight of DNA synthesized in vitro. One possibility is that the enzyme is involved in discontinuous synthesis during normal replication (Masamune et al., 1971). Thus, Okazaki flagments accumulate in polA mutants of E. coli (Kuempel & Veomett, 1970; Okazaki et al., 1971). On the other hand, if DNA polymerase I is important for the efficient joining of Okazaki fragments into high molecular weight DNA in the in vitro T7 system, it is surprising that our in vivo studies with a poIA strain and T7 do not show a similar effect. It is known that polA mutants maintain low levels of DNA polymerase I polymerizing activity and that, in these mutants, the 5' -> 3'

P H A G E T7 DNA R E P L I C A T I O N I N

VITRO: V

555

exonuclease of DNA polymerase I is present at nearly wild-type levels (Lehman & Chien, 1973). I t m a y be that in vivo, but not in vitro, these residual activities are sufficient to allow phage DNA replication to proceed normally. Another role for DNA polymerase I might be to repair gaps in the D N A produced b y non-specific nuclease attack or perhaps as part of normal physiological processes such as transcription or replication. Such limited repair synthesis would be too small to be detected in the pycnographic studies. The fact that we have consistently observed more strand breaks in the template DNA in the absence of DNA polymerase I than in its presence is consistent with such an explanation. The demonstration that DNA molecules the size of an intact chromosome can be synthesized using cell-free extracts suggests that DNA synthesis in the in vitro system m a y closely correspond to in vivo DNA replication in m a n y respects. The accompanying paper (Masker & Richardson, 1976) shows that the fidelity of in vitro D N A replication is sufficiently accurate to produce biologically active DNA molecules. This research was supported by grant no. AI-06045 from the National Institutes of Health, U.S. Public Health Service, and grant no. NP-1D from the American Cancer Society, Inc. One of us (W. E. M.) is a fellow of the Helen Hay Whitney Foundation. The other author (C. C. R.) is the recipient of a Public Health Service Research Career Program Award, no GM-13,634. REFERENCES Center, M. S., Studier, F. W. & Richardson, C. C. (1970). Proc. Nat. Acad. Sci., U.S.A. 65, 242-248. Chamberlin, M. (1974}. J. Virol. 14, 509-516. DeLueia, P, & Cairns, J. (1969). Nature (London), 224, 1164-1166. Grippo, P. & Richardson, C. C. (1971). J. Biol. Chem. 246, 6867-6873. Hanawalt, P. C. & Cooper, P. K. (1971}. In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 21, pp. 221-230, Academic Press, New York and London. Hinkle, D. C. & Richardson, C. C. (1974}. J. Biol. Chem. 249, 2974-2984. Hinkle, D. C. & Richardson, C. C. (1975). J. Biol. Chem. 25{}, 5523-5529. Jovin, T. M., Englund, P. T. & Bertsch, L. L. (1969}. J. Biol. Chem. 244, 2996-3008. Kanner, L. & Hanawalt, P. (1970}. Biochem. Biophys. Res. Commun. 39, 149-155. Kuempel, P. L. & Veomett, G. E. (1970). Biochem. Biophys. Res. Commun. 41, 973-980. Lehman, I. R. & Chien, J. R. (1973} J. Biol. Chem. 248, 7717-7723. Masamune, Y., Frenkel, G. & Richardson, C. C. (1971). J. Biol. Chem. 246, 6874-6879. Masker, W. E. & Richardson, C. C. (1976). J. Mol. Biol. 1{}{},557-567. Miller, J. (1972). Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Modrieh, P. & Richardson, C. C. (1975a}. J. Biol. Chem. 25{}, 5508-5514. Modrich, P. & Richardson, C. C. (1975b). J. Biol. Chem. 250, 5515-5522. Monk, M., Peaty, M. & Gross, J. D. (1971). J. Mol. Biol. 58, 623-630. Moses, R. E. & Richardson, C. C. (1970). Proe. Nat. Acad. Sci., U.S.A. 67, 674-681. Oey, J. L., Str~tling, W. & Knippers, R. (1971}. Eur. J. Biochem. 23, 497-504. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K., Kainuma, R., Sugino, A. & Twatsuki, N. (1968). Cold Spring Harbor Syrup. Quant. Biol. 33, 129-142. Okazaki, R., Arisawa, M. & Sugino, A. (1971}. _Proc. Nat. Acad. Sci., U.S.A. 68, 29542967. Richardson, C. C. (1966). J. Mol. Biol. 15, 49-61. Richardson, C. C., Schildlu'aut, C. L., Aposhian, H. V. & Kornberg, A. (1964). J. Biol. Chem. 239, 222-232. Sadowski, P. D. (1971). J. Biol Chem. 246, 209-216. Sadowski, P. D. & Kerr, C. (1970). J. Virol. 6, 149-155. Str~tling, W. & Knippel~, R. (1973). Nature (London), 245, 195-197. 37

556

W . E . MASKER AND C. C. R I C H A R D S O N

Str~tling, W., Ferdinand, F. J., Krause, E. & Knippers, R. (1973). Eur. J. Biochem. 38, 160-169. Studier, F. W. (1969). Virology, 39, 562-574. Studier, F. W. (1972). Science, 176, 367-376. Wickner, W., Brutlag, D., Schekman, R. & Kornberg, A. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 965-969.