In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation

61

Biochimica et Biophysica Acta, 609 (1980) 6 1 - - 7 4 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 9 9 7 1 0

IN VITRO REPLICATION OF BACTERIOPHAGE T7 DNA DAMAGED BY ULTRAVIOLET RADIATION *

W A R R E N E. M A S K E R and N A N C Y B. K U E M M E R L E

Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) (Received J a n u a r y 2 2 n d , 1980)

Key words: DNA repair; DNA replication; Ultraviolet radiation; (Bacteriophage T7)

Summary The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template. Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the a m o u n t of in vitro DNA synthesis. The residual replication detected when irradiated templates were used was f o u n d to proceed semiconservatively and to result in the production of pieces o f duplex DNA approximately the same size as the average distance between pyrimidine dimers. It was also found that prior irradiation of the template inhibits formation of fast~sedimenting concatemer-like DNA structures normally synthesized in vitro. Hybridization studies demonstrated that the p r o d u c t synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome. This may mean that after ultraviolet irradiation more than one origin of replication exists.

Introduction

In vitro systems offer potentially valuable tools with which to study various aspects of DNA metabolic processes. One such system, developed to study the replication of bacteriophage T7 DNA [1] has proved to be of particular interest since it is capable of using exogenous T7 DNA template to synthesize large amounts of p r o d u c t DNA that has approximately the same molecular weight as

* By a c c e p t a n c e o f this article, the publisher or recipient a c k n o w l e d g e s the U.S. G o v e r n m e n t ' s right to retain a nonexcluslve, r o y a l t y - f r e e license in and to any c o p y r i g h t covering the article.

62 intact T7 genomes [2]. Under normal conditions in vitro reactions synthesize 3 to 10 times more product DNA than the a m o u n t of exogenously supplied template [1,2]. Moreover, the product of the in vitro reaction is fully infective and capable of producing viable T7 phage particles upon transfection of Escherichia coli spheroplasts [3]. A separate in vitro system capable of packaging exogenous T7 DNA has also been described [4] ; and this system has been used to encapsulate the newly synthesized product of the in vitro DNA replication reaction. In this way complete T7 phage particles can be synthesized in a two-step in vitro reaction with an efficiency in excess of 1% viable phage production per genome equivalent of exogenous T7 DNA introduced into the replication system [5]. Recently, it has also been pointed o u t that a significant level of genetic recombination occurs during the in vitro replication process [6]. Collectively these observations argue that the aforementioned in vitro DNA replication system mimics the in vivo situation quite closely and may be used as a meaningful instrument with which to study parameters of DNA replication, recombination, and repair. Our interest in DNA repair mechanisms has p r o m p t e d us to examine the reaction of the in vitro T7 DNA replication system to exogenous DNA template damaged by ultraviolet irradiation. Previous efforts [7,8] have shown that extracts of the type used for in vitro DNA synthesis reactions are deficient at performing incision, the first step of the excision-repair process that removes pyrimidine dimers formed by exposure to ultraviolet radiation [9]. Thus, one advantage of this system is that excision repair mechanisms do not function and quantitative estimates of the response of the DNA replication apparatus to DNA damage can reasonably be based on the assumption that the number of dimers remains constant throughout the reaction. This paper describes the characteristics of the in vitro replication of irradiated DNA and compares results of these measurements under conditions where molecular recombination is reduced by a deficiency in the gene 6 exonuclease. Materials and Methods Strains o f bacteria and bacteriophage T7 Strains of Escherichia coli K-12 used in this study include W3110: thyA sup ° and 011': thyA sup*. Amber mutants of bacteriophage T7 were from the collection of F.W. Studier and include T7 am29 (gene 3), T7 am28 (gene 5) and T7 a m 1 4 7 (gene 6). In the text the phage mutants are designated by subscript indicating the defective gene. Thus a T7 am29, am147 m u t a n t is written as T73,6. Media, chemicals and reagents Sources of materials for media and reaction mixtures have been described [3,5,8]. Agarose (Type II, medium EEO), ethidium bromide, and bromocresol green were obtained from Sigma. Restriction endonuclease EcoRI was purchased from N e w England Biolabs, Beverly, MA. A damage-specific endonuclease from Micrococcus luteus was a gift from W.L. Carrier [10]. DNA was

63 prepared as described by Richardson [11]. Amounts of DNA are expressed as nucleotide phosphorous equivalents or, in some cases, as T7 genome equivalents where 1 nmol of DNA is taken to equal 7.5 • 109 intact chromosomes of T7 phage.

Reaction conditions for in vitro DNA synthesis DNA synthesis reactions were performed using extracts from bacteria infected with T73 or T73,6 as previously described [1,5]. In these experiments 3 nmol of DNA, either irradiated or undamaged, was included in the reaction. Irradiation of DNA was usually carried out at a concentration of 1.0 to 1.3 mM. DNA was irradiated at room temperature 0.1 ml at a time with constant stirring by exposure to a pair of General Electric germicidal lamps with an incident dose rate of 1.6 J / m 2 per s. Under these conditions 1 J / m 2 introduces 0.46 pyrimidine dimers per T7 genome equivalent of DNA [8]. DNA synthesis reactions were carried out at 30°C with incubation times of 10 to 40 min. To measure total DNA synthesis, reactions which included [32p]dATP were stopped by the addition of ice-cold 1 N HC1-0.1 M PPi and acid-insoluble radioactivity was determined. For analysis by zone sedimentation or isopycnic gradients DNA synthesis was terminated by the addition of 0.01 ml of 0.5 M EDTA per 0.1 ml reaction mixture. The material prepared for sucrose gradient analysis was incubated for 10 min at 42°C with an equal volume of 20 mM Tris-HC1 (pH 7.5), 1 M NaC1 and 4% (w/v) Sarkosyl before being layered onto the gradient.

Sedimentation analysis Methods used for sedimentation through neutral and alkaline sucrose and for isopycnic analysis were the same as previously described [5].

Restriction endonuclease digestion 3H-Labeled DNA was prepared from T7s phage ( 3 4 0 0 0 cpm//~g). Two hundred micrograms of labeled or unlabeled T7s DNA was incubated at 37°C in 100 mM Tris (pH 7.5) and 10 mM MgSO4 in the presence of 400 units of restriction endonuclease EcoRI in a total volume of 11.7 ml for 3 h [12]. The reactions were terminated with EDTA to a concentration of 20 mM. Samples were concentrated by ethanol precipitation or drying under a stream of air. Reactions went to completion, as evidenced by failure to detect uncleaved DNA on 0.75% agarose horizontal slab gels; this procedure would have revealed uncleaved DNA in amounts less than 5% of the input DNA.

Resolution of restriction fragments using gel electrophoresis The two fragments resulting from digestion of T7s DNA by EcoRI, which cleaves the DNA 46% from the genetic left end [12] were approx. 18 400 and 21 600 base pairs in length. These fragments could be resolved on an 0.8% agarose 30 mm horizontal slab gel using Buffer A (Buffer A is 72 mM Tris, 34 mM Na2HPO4, 26 mM NaH2PO4, 2 mM EDTA, adjusted to pH 7.5 [13]). After electrophoresis at 1 V/cm for 21 h, distances of migration relative to the buffer

64 f r o n t were 0.14, 0.12, and 0.07 for the small fragment, large fragment, and uncleaved DNA, respectively. Preparative-scale separation of 200 /~g quantities of EcoRI-cleaved T7s DNA was achieved on a 2 cm × 37 cm 0.75% agarose tube gel. A flat meniscus was obtained by floating a 2 cm teflon disc atop the gel until solidification; gels were pre-run in Buffer A at 75 V for 4--12 h prior to addition of sample. Samples were adjusted to 17% glycerol (v/v) by addition of a solution of 0.05% bromocresol green in 50% glycerol, layered onto the gel, and electrophoresed at 75 V for approx. 36 h, or until the dye front approached the b o t t o m of the gel; buffer was recirculated during the electrophoresis and the temperature was maintained by circulating water through the column jacket. DNA bands were localized by staining the surface of the gel with a minimum volume of ethidium bromide at a concentration of 1 /~g/ml and subsequently separated with a sharp razor blade. Each band was sliced into 1 mm sections, and DNA was harvested by the freeze-squeeze m e t h o d [14]. Each fragment was recovered in approx. 45% yield at a concentration of 7--9 ~g/ml.

Hybridization The procedure employed for DNA-DNA hybridization was essentially that of Fujimura [15]. DNA to be immobilized on filters was denatured at a concentration of < 1 0 / ~ g / m l in 0.1 N NaOH at room temperature for 15 min. After neutralization with HC1, the DNA solution was adjusted to the concentration of Buffer B (Buffer B is 0.9 M NaC1 and 0.09 M sodium citrate, pH 7). Nitrocellulose membranes (Schleicher and Schuell B-6, 25 mm) soaked in Buffer B were loaded with 12 /~g of the denatured DNA fragments obtained from the agarose gels or from T7 wild-type phage; blanks were likewise prepared using salmon sperm DNA. All filters were rinsed with 30 ml Buffer B, dried at room temperature overnight and at 80°C for 3 h. DNA to be hybridized to the DNA immobilized on the membranes was obtained from DNA synthesis reactions. DNA synthesis was allowed to proceed for 20 min in the presence of [32P]dATP as described above in three 1-ml reaction mixtures in the absence of exogenously added template and in the presence of unirradiated DNA or DNA which had received 15 J / m 2 of ultraviolet radiation (sufficient to introduce about 7 dimers per chromosome). Portions of each reaction mixture were removed for alkaline sucrose gradient analysis and quantitation; the remainder was extracted with phenol and dialyzed against several changes of 10 mM Tris (pH 7.5)--0.1 mM EDTA. Samples were acid-precipitated, filtered, and radioactivity determined to quantitate 3H-template and 32P-product DNA. DNA to be hybridized to membrane-bound DNA was denatured in 0.1 N NaOH, neutralized, and sonicated for 1 min with a Bronwill sonicator at o u t p u t of 75 at the resonant frequency. Samples were adjusted to Buffer C (Buffer C is 0.9 M NaC1, 0.09 M sodium citrate, pH 7, 40% (v/v) formamide). Denatured, sonicated unlabeled DNA in Buffer C was added to the DNA synthesized from an unirradiated template and to that synthesized in the absence of exogenously added template such that the ratio of template + added DNA to 32p-product DNA was constant. DNA-containing filters were soaked for 10 min in Buffer C, then placed in

65 1-dram glass vials, and 0.5 /~g of the appropriate DNA solutions was added to each vial; volume was brought to 4.0 ml using Buffer C. Incubation was at 38°C for 20 h with shaking in a water bath. Each filter was rinsed twice in Buffer C and 3 times in 3 mM Tris (pH 9.3), dried, and counted in toluene-based scintillation fluid. The solution remaining in each incubation vial was removed and, after rinsing the vial, precipitated with 2 ml of 20% cold trichloroacetic acid. Precipitates were collected on nitrocellulose filters, rinsed twice with 5% trichloroacetic acid and once with 95% ethanol, dried, and radioactivity determined. The sum of 32p activity b o u n d to the filter and that which remained unhybridized in solution was approx. 100%. All samples were determined in duplicate; linearity of binding had been established for filters loaded with 12 ~g DNA in the range 0.5--2.0 ~g of added [14C]DNA (data not shown). Results

The effect o f damage on replication o f a linear duplex DNA synthesis performed by extracts from T 7 r i n f e c t e d bacteria was compared with that performed by recombination deficient [6,16] extracts from T73.6-infected cells. The results, shown in Fig. 1, indicate that with either extract DNA synthesis performed with irradiated templates is nearly linear with time for up to 40 rain and that there is a strong decrease in synthesis with .increasing ultraviolet-irradiation dose. This figure also shows residual replication from endogenous DNA carried during infection into the E. coli used to prepare the extracts [2]. Presumably, the synthesis detected in the absence of any exogenous DNA represents the maximum amount of replication of endogenous DNA that can occur in this system since it takes place w i t h o u t competition from exogenously added template. Thus far, we have been unable to completely remove or inactivate the contaminating endogenous DNA w i t h o u t adversely affecting the properties of the in vitro replication. Therefore, we have restricted our analysis to doses less than 25 J / m 2 where the contribution due to endogenous DNA does n o t present a serious problem and, in Figs. l c and l d , have corrected for the presence of endogenous DNA. These data indicate a rapid fall-off of DNA synthesis with increasing dose and show a close similarity between results found using extracts from T73- or T73,6-infected cells. Although prior irradiation of the template causes a reduction in the rate of in vitro DNA synthesis, close to 50% of the maximum rate of synthesis persists even at a dose of 20 J / m 2, which is sufficient to introduce a b o u t 10 pyrimidine dimers per genome [8]. This a m o u n t of synthesis is more than what would be expected if each pair of replication forks advanced from a single origin of replication until their progress was halted by a pyrimidine dimer. Sedimentation analysis Sedimentation analysis was used to help characterize the product synthesized in vitro after irradiation of the input DNA. The profiles of product and template DNAs recovered after in vitro DNA synthesis and sedimentation under denaturing conditions are shown in Fig. 2.

66 i

i

I

i

i

i

b

Cl

50

/ / / / /

0

E 20

f

Z

/

oY

/ /

/

o/

I0

0 I

1O0

20

30

F

,

,

40

TIM

lO

(min)

/ //*

/

i

10

/

/•

/

20

•

/// •~ f

50

4'0

6'0

80

i

C

80

60

\

\ \ \•

\ 0

Z

'-" 4 0

\

20

0o

2b

\

\

\

\\• \

o..<__ 4'0 ~b

4:> ~oo

L~

4o

1O0

ULTRAVIOLET DOSE (J/m 2) Fig. 1. E f f e c t o f u l t r a v i o l e t irradiation o n k i n e t i c s o f D N A s y n t h e s i s in T 7 3 - and T 7 3 , 6 - i n f e c t e d e x t r a c t s . W i l d - t y p e T 7 D N A w h i c h had b e e n irradiated w i t h u l t r a v i o l e t - r a d i a t i o n f l u e n c e s o f 0, 25, or 5 0 J / m 2 w a s a d d e d t o e x t r a c t s f r o m T 7 3 - and T 7 3 , 6 - i n f e c t e d strain W 3 1 1 0 and i n c u b a t e d at 3 0 ° C u n d e r standard c o n d i t i o n s for D N A s y n t h e s i s as d e s c r i b e d in Materials and M e t h o d s . [ 3 2 p ] d A T P was p r e s e n t at 2 . 0 c p m / p m o l . F r a m e s (a and b): A t the i n d i c a t e d t i m e s , 50-~tl a l i q u o t s o f e a c h r e a c t i o n m i x t u r e w e r e r e m o v e d a n d acid p r e c i p i t a t e d . R a d i o a c t i v i t y w a s d e t e r m i n e d and a m o u n t o f D N A s y n t h e s i z e d w a s c a l c u l a t e d . o ©, n o u l t r a v i o l e t radiation; ~ A 25 J/m2; B a, 5 0 J / m 2 ; 0 0, n o e x o g e n o u s D N A a d d e d t o t h e r e a c t i o n . F r a m e a: e x t r a c t f r o m T 7 3 - i n f e c t e d cells; F r a m e b: e x t r a c t f r o m T 7 3 , 6 - i n f e c t e d cells. T h e l o w e r f r a m e s s h o w t h e p e r c e n t r e m a i n i n g D N A s y n t h e s i s as a f u n c t i o n o f u l t r a v i o l e t - r a d i a t i o n d o s e . As a b o v e , D N A w h i c h h a d b e e n irradiated w i t h t h e i n d i c a t e d d o s e s w a s a d d e d t o standard r e a c t i o n m i x t u r e s . A f t e r 2 0 m i n i n c u b a t i o n , a l i q u o t s w e r e r e m o v e d and acid p r e c i p i t a t e d . A f t e r s u b t r a c t i o n o f t h e a m o u n t o f s y n t h e s i s resulting f r o m e n d o g e n o u s D N A , t h e a m o u n t o f D N A s y n t h e s i z e d at e a c h d o s e w a s c o m p a r e d t o t h e u n i r r a d i a t e d c o n t r o l . F r a m e c: o o e x t r a c t s f r o m T 7 3 - i n f e c t e d cells, F r a m e d: • ...... • , e x t r a c t s f r o m T 7 3 , 6 - i n f e c t e d cells.

Interpretation of these profiles must include a consideration of the fact that the molecules recovered from the reaction are near to genome size and that the newly synthesized product will, on the average, be of shorter length than the template. As is apparent from Fig. 2, D N A synthesis performed with the unirradiated template resulted in the generation of about three times as much product as template. The contribution of partially replicated molecules, produced during second and third rounds of replication [2], results in an ensemble of product molecules with an overall molecular weight slightly smaller than that of the template. Application of 10--20 J/m 2 resulted in newly synthesized molecules with a length comparable to the product synthesized from template endogenous to the extract (with no exogenous T7 D N A added to the reaction). To compare the size of the product D N A with the average spacing between pyrimidine dimers the D N A recovered after in vitro D N A

67

400

,

me

, CONTROL

,9~' i/~

o

300 t

2ooF

r

~, ! 2!

3OO/b

i

10 J/m ~ /x

_~ 2oo o

~"t,

E {3,, < 1OO z,'m

o

c

20 J/m 2

,oo 2ootd 100~

8ottom

2

No DNA

/

/ ~

4O

FRACTION NO.

Top

Fig. 2. C h a r a c t e r i z a t i o n b y z o n a l c e n t r i f u g a t i o n o n a l k a l i n e s u c r o s e g r a d i e n t s o f D N A s y n t h e s i z e d b y e x t r a c t s f r o m T 3 , 6 - i n f e c t e d cells. S t a n d a r d r e a c t i o n m i x t u r e s f o r D N A s y n t h e s i s c o n t a i n i n g e x t r a c t s f r o m T 7 3 , 6 - i n f e c t e d cells w e r e i n c u b a t e d f o r 2 0 r a i n a t 3 0 ° C w i t h w i l d - t y p e T 7 3 H - l a b e l e d D N A a t 7 . 6 c p m / p m o l w h i c h h a d r e c e i v e d (a) n o i r r a d i a t i o n ; (b) 1 0 J / m 2 ; a n d (c) 2 0 J / m 2 o f i r r a d i a t i o n ; r e a c t i o n m i x t u r e (d) c o n t a i n e d b u f f e r ( 1 0 m M Tris ( p H 7 . 5 ) , 0 . 1 m M E D T A ) i n p l a c e o f D N A . [ 3 2 p ] d A T P w a s p r e s e n t a t 8.1 c p r n / p r n o l . R e a c t i o n s w e r e t e r m i n a t e d b y a d d i t i o n o f E D T A t o a c o n c e n t r a t i o n o f 5 0 rnM a n d i n c u b a t e d w i t h Tris, NaC1, s a r k o s y l 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 ; 0 . 1 m l a l i q u o t s w e r e l a y e r e d o n t o 5---20% a l k a l i n e s u c r o s e g r a d i e n t s . C e n t r i f u g a t i o n in a S p i n c o S W 5 6 r o t o r w a s a t 4 9 0 0 0 r e v . / m i n f o r 2 h. S i x - d r o p f r a c t i o n s w e r e c o l l e c t e d , a c i d p r e c i p i t a t e d , a n d c o l l e c t e d o n t o W h a t m a n G F / C filters. R a d i o a c t i v i t y w a s d e t e r m i n e d ; p r o f i l e s are s h o w n , o o, 3 H - l a b e l e d t e m p l a t e ; ~- . . . . . ~ 3 2 p . labeled product. The arrow indicates the position of intact T7 DNA sedimented under identical conditions.

synthesis was treated with an endonuclease specific for pyrimidine dimers and then sedimented through alkaline sucrose. The profiles shown in Fig. 3 indicate that the product has a slightly smaller size than the average interdimer distance. A comparison between Fig. 3 and Fig. 2C, which shows D N A taken from the same experiment, indicates that the product D N A which was subjected to endonuclease treatment (Fig. 3) has essentially the same molecular weight distribution as the product D N A in Fig. 2C which was n o t exposed to the endo-

68 i

i

15C

8,.~--~-L

,

\

~-- 10C 12L 7 123

5C

o

\ p i j,

,'

& od //

?/

O"0 0 0-0 0/0 0-0 I

O0

10

20

50

40

FRACTION NOI Fig. 3. C o m p a r i s o n of size of product DNA with interdimer distance. A reaction mixture containing an extract from T73,6-infected c e l l s a n d D N A i r r a d i a t e d w i t h 20 J / m 2 w a s i n c l u d e d in t h e e x p e r i m e n t d e s c r i b e d i n F i g . 2. R e a c t i o n c o n d i t i o n s w e r e i d e n t i c a l w i t h t h o s e in Fig. 2 e x c e p t t h a t t h e r e a c t i o n w a s terminated by the addition of EDTA to a concentration o f 20 raM. A n e n d o n u e l e a s e f r o m M. l u t e u s t h a t is s p e c i f i c f o r u l t r a v i o l e t - r a d i a t i o n d a m a g e w a s a d d e d t o t h e r e a c t i o n m i x t u r e as p r e v i o u s l y d e s c r i b e d [ 8] and incubation at 3 0 ° C w a s c o n t i n u e d f o r a n a d d i t i o n a l 10 m i n . P r e v i o u s e x p e r i m e n t s [ 8 ] h a v e s h o w n that the endonuclease is a c t i v e u n d e r t h e s e c o n d i t i o n s a n d p r o d u c e s o n e s i n g l e - s t r a n d b r e a k p e r p y r i m i d i n e d i m e r . E D T A w a s a d d e d t o raise t h e f i n a l c o n c e n t r a t i o n t o 50 m M b e f o r e t r e a t m e n t w i t h Tris, NaC1 and sarkosyl followed by sedimentation t h r o u g h a l k a l i n e s u c r o s e as d e s c r i b e d i n Fig. 2. P r o f i l e s o f r a d i o a c t i v i t y are s h o w n , o o, 3 H - l a b e l e d t e m p l a t e ; A . . . . . . ~, 3 2 p - l a b e l e d p r o d u c t . T h e a r r o w i n d i c a t e s the position of intact T7 DNA sedimented under these conditions.

nuclease. Therefore, no significant number of pyrimidine dimers were recombinationaUy transferred to the product. The evidence (Fig. 3) that the product DNA is on the average slightly smaller than the interdimer distance argues against any large-scale replicative by-pass such as might occur if misinsertion of bases across from the pyrimidine dimers were taking place. Neutral sucrose sedimentation analysis (Fig. 4) confirmed that when unirradiated DNA is employed as a substrate most of the product has a double-strand molecular weight close to that of intact T7 genomes. Applications of ultraviolet radiation to the template caused a marked reduction in the overall doublestrand molecular weight of the newly synthesized DNA. However, these profiles show no reduction in the molecular weight of the template. Also, it is seen that the component of the product which is of the same molecular weight as the template has not been totally eliminated by irradiation. A comparison with Fig. 2 demonstrates that the conditions used here produce no single-strand interruptions in the template. Thus, the most straightforward interpretation of the data in Fig. 4 suggests that the large amount of double-stranded lowmolecular-weight DNA, seen to be approximately the same size as the interdimer-distance, probably arises from re-initiation of DNA synthesis at one or more internal origins followed by separation of the newly synthesized material

69 400 a

!

CONTROL

,

300 20C

#

x

10C

0 30C

b

10 J / m 2

20C

:5, (]

E

,

,

,

"e'

C'NTROL I 0 I

x

,~ IOC

2

Z

c

:

'T

,,

/

olJ, ~,\ 200[d

%

01 ~ ,

21b

;

°/%d 'd'

~-~*~

,

'

~°, ~ a ~

,

HH

~ LL

10 J / m 2

No DNA II

-20

Bottom

/

FRACTION NO,

Top

o-,~

30

~

'

40

"

50

NORMALIZED FRACTION NO.

Fig. 4. C h a r a c t e r i z a t i o n b y z o n a l c e n t r i f u g a t i o n o n n e u t r a l s u c r o s e g r a d i e n t s o f D N A s y n t h e s i z e d b y e x t r a c t f r o m T 7 3 6 - i n f e c t e d cells. S t a n d a r d r e a c t i o n m i x t u r e s f o r D N A s y n t h e s i s w e r e i n c u b a t e d f o r 2 0 m i n a t 3 0 ° C w i t h ' 3 H - l a b e l e d D N A a t 7.6 c p m / p m o l w h i c h h a d b e e n i r r a d i a t e d w i t h d o s e s o f (a), 0, (b) 1 0 , o r (c) 2 0 J / m 2 ; a r e a c t i o n m i x t u r e (d) c o n t a i n i n g n o e x o g e n o u s D N A w a s i n c u b a t e d u n d e r i d e n t i c a l c o n d i t i o n s . [ 3 2 p ] d A T P w a s p r e s e n t a t 7 . 8 c p m / p m o L As d e s c r i b e d i n t h e l e g e n d t o Fig. 2, r e a c t i o n s w e r e t e r m i n a t e d b y a d d i t i o n o f E D T A a n d i n c u b a t e d w i t h Tris, NaCI, s a r k o s y l ; 0 . 1 m l p o r t i o n s w e r e l a y e r e d o n t o 5 - - 2 0 % n e u t r a l s u c r o s e g r a d i e n t s w i t h a 0 . 2 m l s h e l f o f 8 4 % s u c r o s e . C e n t r l f u g a t i o n in a S p i n c o S W 5 6 r o t o r w a s a t 4 9 0 0 0 r e v . / m i n f o r 2 h. F r a c t i o n s w e r e c o l l e c t e d , a c i d p r e c i p i t a t e d , a n d r a d i o a c t i v i t y was determined, o o, 3 H - l a b e l e d t e m p l a t e ; ~- . . . . . ~, 3 2 p - l a b e l e d p r o d u c t . T h e a r r o w i n d i c a t e s t h e position of intact T7 DNA sedimented under identical conditions. Fig. 5. I s o p y c n i c g r a d i e n t a n a l y s i s o f D / q A s y n t h e s i z e d i n v i t r o f r o m u n i r r a d i a t e d a n d i r r a d i a t e d t e m p l a t e s . T w o O.S-ml r e a c t i o n m i x t u r e s c o n t a i n i n g a n e x t r a c t o f T 7 3 6 - i n f e c t e d s w a i n W 3 1 1 0 w e r e i n c u bated for 20 rain under standard conditions for DNA synthesis. [32p]'dATP was present at 8.9 epm/pmol. (a) c o n t a i n e d 9 n m o l o f w i l d - t y p e T 7 ( 3 H , I 3 C , I S N ) - l a b e l e d DN'A a t 3 . 9 e p m / p m o l . (b) c o n t a i n e d 9 n m o l of the same DNA which had been irradiated with 10 J/m 2 of ultraviolet radiation. The reactions were t e r m i n a t e d a n d t h e prod~act a n a l y z e d b y I s o p y c n l c e e n t r i f u g a t i o n as d e s c r i b e d { 5 ] . A c i d p r e e i p i t a b l e material was collected and radioactivity was determined, o o, [ 3 H , I 3 C , I S N ] D N A ; ~- . . . . . A 3 2 p . labeled product.

(with both strands 32P-labeled) when the replication fork meets a gap caused by an encounter with a pyrimidine dimer during the first round of replication. Alternatively, a replication fork produced by a second or third initiation event

70 is likely to generate short duplex DNA fragments upon collision with a preceding replication fork whose progress was stalled by a dimer in its path. The product of in vitro DNA synthesis was also examined with isopycnic gradients. (3H,13C,1SN)-Labeled T7 DNA was either irradiated with 10 J/m 2 or left unirradiated before being incubated in the standard in vitro DNA synthesis reaction. The profiles recovered after sedimentation in CsC1 are shown in Fig. 5. The upper frame (a) is typical of semiconservative replication of undamaged DNA as reported in earlier studies [3,5]. The lower frame (b) shows limited transfer of the heavy DNA to the hybrid position. In fact a b o u t 40-50% of the template is associated with light-density product, probably by hydrogen bonding. This suggests that ultraviolet-irradiated template molecules are only incompletely copied during the first round of replication. Most of the p r o d u c t is of light-light density. Measurements of DNA synthesis in the absence of exogenous template (carried o u t as part of this experiment) show that a m a x i m u m of 35% of the light-light 32P-labeled DNA in Fig. 5B may have been synthesized from endogenous template. The remainder of the light-density material was apparently synthesized by multiple rounds of replication using the irradiated DNA as the initial template. Collectively the sedimentation profiles in Figs. 2--5 reinforce the notion that multiple initiation events take place on the same DNA molecule and that short duplex DNA pieces are generated upon separation from the parent molecule.

Hybridization experiments If most of the DNA synthesized from an irradiated template containing at least 6 dimers is formed by replication forks initiated at the primary origin located 17% from the left end of the T7 DNA molecule [17] it is expected that the p r o d u c t DNA would contain base sequences which would cause preferential base pairing with sequences found on the left end of the T7 chromosome. If, on the other hand, ultraviolet radiation stimulates initiation at secondary origins, product DNA could arise from all portions of the DNA molecule and would exhibit affinities for both the right and left fragments as is the case for the unirradiated sample which has undergone more than one complete round of replication. A DNA-DNA hybridization experiment was performed to test this. T7 DNA with an am28 mutation (gene 5) was digested with EcoRI endonuclease so as to produce a single cleavage site located 46% from the genetic left end [12]. Fragments from each end of the chromosome were b o u n d to nitrocellulose filters and used for hybridization experiments. 3H-Labeled wild-type T7 DNA was irradiated and used as template in an in vitro DNA synthesis reaction. As seen in Table I, the 32P-labeled p r o d u c t synthesized from irradiated template showed only a very slight preference for hybridization to the left end of the chromosome, and a significant a m o u n t of DNA produced after in vitro replication of the irradiated template shows base h o m o l o g y with the right end of the DNA molecule. This also argues in favor of multiple initiation sites and favors the hypothesis that some initiation events occur on the right side of the chromosome. The effect o f the gene 6 exonuclease The experiments presented above were performed using extracts from

71 TABLE I DNA-DNA HYBRIDIZATION D N A f r o m t h e l e f t e n d , r i g h t e n d , o r e n t i r e T 7 c h r o m o s o m e w a s i m m o b i l i z e d o n n i t r o c e l l u l o s c filters as described in Materials and Methods. A standard DNA synthesis reaction was performed using an extract from T73,6-infected strain W3110 and 3H-labeled T7 DNA that was either unirradiated or exposed to 15 J / m 2 o f u l t r a v i o l e t r a d i a t i o n . T h e p r o d u c t D N A w a s l a b e l e d w i t h [ 3 2 p ] d A T P . In t h i s e x p e r i m e n t , 1 0 . 8 n m o l o f D N A w a s s y n t h e s i z e d in t h e 0 . 1 m l c o n t r o l r e a c t i o n u s i n g u n t r r a d i a t e d D N A , a n d s y n t h e s i s d r o p p e d t o 7.1 n m o l w h e n t h e t e m p l a t e c o n t a i n e d d a m a g e . T h e ' b a c k g r o u n d ' d u e t o e n d o g e n o u s D N A was 1.5 nmol. After extraction with phenol to remove protein the DNA was dialyzed extensively and samp l e s c o n t a i n i n g a t o t a l o f 0 . 5 /~g D N A w e r e h y b r i d i z e d w i t h t h e D N A i m m o b i l i z e d o n t h e filter 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 . H y b r i d i z a t i o n w a s p e r f o r m e d u n d e r c o n d i t i o n s w h e r e t h e a m o u n t o f D N A h y b r i d i z e d w a s l i n e a r w i t h t h e a m o u n t o f D N A p r e s e n t in t h e h y b r i d i z a t i o n r e a c t i o n . T h e a m o u n t o f 3 2 p - l a b e l e d D N A b o u n d t o t h e filter w a s d e t e r m i n e d a n d c o m p a r e d w i t h t h e a m o u n t o f a c i d - i n s o l u b l e 32p-labeled DNA added to the hybridization reaction mixture (input DNA) to calculate the percent of i n p u t D N A b o u n d t o filter. T h e p r o c e d u r e is d e t a i l e d i n M e t h o d s . ' B l a n k s ' , filters o n t o w h i c h 2 0 / ~ g d e n a tured salmon sperm DNA had been immobilized, were incubated with one of each of the duplicate samples a n d b o u n d less t h a n 0 . 0 4 % o f t h e i n p u t D N A . C o n t r o l s i n w h i c h r i g h t - e n d a n d l e f t - e n d f r a g m e n t s w e r e h y b r i d i z e d w i t h t h e o p p o s i t e f r a g m e n t i m m o b i l i z e d o n t h e filter also s h o w e d n e g l i g i b l e h y b r i d i z a t i o n . A d d i t i o n a l c o n t r o l s i n d i c a t e d t h a t c r o s s h y b r i d i z a t i o n b e t w e e n left- a n d r i g h t - e n d f r a g m e n t s w a s negligible, that DNA replicated from urdrradiated or irradiated templates bound to whole T7 DNA with nearly equal efficiency (about 30%), and that the DNA synthesized from endogenous template had equal affinities for the left and right ends of the T7 chromosome. Sample

Description D N A i m m o b i l i z e d o n filter:

A B C D

No irradiation 15 J/m 2 No exogenous DNA Whole T7 DNA

Percent of product bound Left end

Right end

Whole

23.1 24.0 16.5

25.8 21.0 18.2

32.6 29.2 18.1 23.0

T73.6-infected strain W3110. Previous studies [5] have shown that when in vitro DNA synthesis is performed by similar extracts prepared from T73infected cells very complex concatemer-like DNA structures are formed. Moreover, we have shown that a significant level of genetic recombination takes place during in vitro DNA synthesis performed by extracts from T73infected cells whereas this recombination is about 20-fold lower when measured using extracts from bacteria infected with T73,6 [6]. In view of those observations it was possible that when T73-infected cells were used as the source of extract, recombination between DNA molecules containing pyrimidine dimers might produce damage-free structures which could be candidates for preferential in vitro replication. An experiment similar to the one shown in Fig. 2 was performed with extracts from T73-infected strain W3110. Profiles recovered from alkaline sucrose gradient analysis are shown in Fig. 6. Prior irradiation of the template appears to have less effect on the size of the product when.extracts containing normal levels of the gene 6 product are employed. However, it appears that irradiation causes a reduction in the amount o f newly synthesized DNA with a single-strand molecular weight equal to that of a complete T7 genome. Thus, even though recombination might produce enough damage-free molecules to show improved biological activity after irradiation [6], recombinational exchanges of DNA molecules do not completely restore the high-molecular-weight component of the product DNA.

72 200

i

i

0

400

i

i~

150

CONTROL

CONTROL

500

150

xxx , , ¢ ~i

450S

100 2OC

%

1OO

t

% 50

X

\o

i

, o E

50

10(

. '~'

O

i

i

~-

150

%

O

b

1

E

10 J/m 2

10 U-/rn 2

500

9 /h

o

lOO c~ E

v

v

2OO

<

g

d

q

\

5O

100

..

50 A4

O

,~

O

5OO c

100

150

C 20 J/m 2 A

20 J i m 2

100 2OO

50

0

Bottom

50

100

/ 0

,%o%

.

10

20

30

FRACTION NO,

40

Top

O 0

i

10

20

30

Bottom

40

o

Top

FRACTION NO,

Fig. 6. C h a r a c t e r i z a t i o n o f D N A s y n t h e s i z e d b y e x t r a c t s f r o m T 7 3 o i n f e c t e d cells. D N A s y n t h e s i s r e a c t i o n s e m p l o y i n g a n e x t r a c t p r e p a r e d f-tom T 7 3 - i n f e c t e d s t r a i n W 3 1 1 0 w e r e c a r d e d o u t as d e s c r i b e d in t h e l e g e n d t o Fig. 3 w i t h t e m p l a t e s w h i c h h a d r e c e i v e d (a) 0, (b) 10, o r (c) 20 J / m 2 o f u l t r a v i o l e t r a d i a t i o n . [ 3 2 p ] d A T P w a s p r e s e n t at 11.6 c p m / p m o l . C e n t r i f u g a t i o n on alkaline s u c r o s e g r a d i e n t s w i t h 0.2 m l 6 0 % a l k a l i n e s u c r o s e s h e l f was a t 4 9 0 0 0 r e v . / m i n f o r 2 h. A c i d - p r e c i p i t a b l e m a t e r i a l was c o l l e c t e d t o o b t a i n t h e profiles s h o w n , o o, 3 H - l a b e l e d t e m p l a t e ; A. . . . . . z~ 3 2 p . l a b e l e d P r o d u c t . T h e a r r o w i n d i c a t e s the position of intact T7 DNA. Fig. 7. C h a r a c t e r i z a t i o n o n n e u t r a l s u c r o s e g r a d i e n t s o f D N A s y n t h e s i z e d b y T 7 3 - i n f e c t e d e x t r a c t . D N A s y n t h e s i s r e a c t i o n s e m p l o y i n g a T 7 3 - i n f e c t e d e x t r a c t w e r e c a r r i e d o u t as d e s c r i b e d in t h e l e g e n d t o Fig. 3 w i t h t e m p l a t e s w h i c h h a d r e c e i v e d (a) 0, (b) 10, or (c) 20 J / m 2 o f u l t r a v i o l e t r a d i a t i o n . [ 3 2 p ] d A T P w a s p r e s e n t a t 5.3 c p m / p m o l . C e n t r i f u g a t i o ~ o n n e u t r a l s u c r o s e g r a d i e n t s w i t h 0.2 m l s h e l f o f 84% s u c r o s e w a s a t 2 0 0 0 0 r e v . / m i n f o r 4 5 rain in a S p i n c o SW56 r o t o r . A c i d - p r e c i p i t a b l e m a t e r i a l was c o l l e c t e d t o obtain the profiles shown, o o, 3 H - l a b e l e d t e m p l a t e ; A. . . . . . a 3 2 p . l a b e l e d p r o d u c t . A r r o w s i n d i c a t e t h e p o s i t i o n s o f 3 H - l a b e l e d T 7 p h a g e ( 4 5 0 S) a n d T 7 [ 3 H ] D N A (36 S).

We also examined the effect of ultraviolet irradiation on the production of fast-sedimenting concatemer-like structures normally found after in vitro DNA synthesis performed by extracts from T73-infected cells. To do this an experiment like the one in Fig. 6 was performed, and neutral sucrose gradients were used to examine the product. As seen in Fig. 7, application of ultraviolet

73 radiation to the template causes a marked reduction in the fast-sedimenting component of DNA. This suggests that production of concatemers is effectively blocked when the template molecules contain pyrimidine dimers. Alternatively, ultraviolet-induced reduction of the number of recombinant molecules may lower the sedimentation rate. Discussion

In vitro host-cell reactivation experiments have shown that sensitivity to ultraviolet radiation was increased by about one order of magnitude when DNA replication occurred under conditions which lowered recombination [6]. Under conditions where normal recombination takes place pyrimidine dimers constitute a formidable barrier to DNA replication (Fig. 1). However, even at a relatively high dose of 20 J/m 2 where the presence of about 10 dimers per genome lowers survival to 5% of the unirradiated control [4,6] DNA synthesis remains at about 40% of the value found with unirradiated template. This residual synthesis exceeds the amount of template included in the reaction and might have arisen of preferential replication of damage-free molecules or from partial replication and undamaged regions of the genome. Recombination could serve to improve survival by postreplication repair [18,19] or by multiplicity reactivation [20]. Our sedimentation data suggest that even under conditions that allow recombination to take place only a small number of the DNA molecules synthesized in vitro from irradiated template are of intact genome size. However, recombination between the duplex DNA fragments or between parent molecules with replication forks stalled at pyrimidine dimers may very well contribute to an improved yield of biologically active DNA molecules. Recently, other workers have investigated in vivo replication and recombination of ultraviolet-irradiated bacteriophage T7 with the intent of testing the hypothesis that recombination occurs primarily between partial replicas of the damaged genome [21,22,23]. Their data suggest a primary origin near the left end of the molecule but allow other origins scattered along the left 30% of the genome [21,23]. Hybridization studies carried out with the product of in vivo replication of relatively heavily irradiated T7 DNA show that the newly synthesized material hybridizes primarily to restriction fragments obtained from the left end of the chromosome [21]. However, at lower doses (somewhat higher than the dose used in the present work) product DNA did show base homology with restriction fragments from the right end of the T7 genome [21]. A possible explanation for this result is the existence of one or more secondary origins located somewhere on the right half of the T7 genome. It may be that available origins of replication are not used with equal probability and that contributions from some of the alternative initiation sites are difficult to detect when DNA synthesis 'is drastically r e d u c e d b y heavy irradiation. However, the data in Table I, which are based on a dose sufficient to introduce about 7 dimers per genome and result in a survival of approx. 8%, are most oompatible with the presence of a second replicative origin either right of gene 5 or sufficiently close to gene 5 to allow replication of DNA right of that marker. Other in vitro experiments [24] have revealed three replicative origins located 18%,

74 31% and 86% from the left end of the chromosome. The availability of these initiation sites may figure prominently in the results displayed in Table I. Also, it must be remembered that initiation at secondary (perhaps nonspecific) origins might be stimulated after ultraviolet irradiation, possibly as the result of hesitation of the replication apparatus at the barriers imposed by pyrimidine dimers. Our data indicate that the primary component of the product generated during in vitro replication of ultraviolet-irradiated template consists of duplex DNA molecules approximately the same length as the interdimer distance. We favor the interpretation that this DNA synthesis reaction proceeds by multiple initiation events occurring at more than one origin of the T7 chromosome.

Acknowledgment This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. References 1 2 3 4 5 6

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Hinkle, D.C. and Richardson, C.C. (1974) J. Biol. Chem. 249, 2974--2984 Masker, W.E. and Richardson, C.C. (1976) J. Mol. Biol. 100, 543--556 Masker, W.E. and Richardson, C.C. (1976) J. Mol. Biol. 100, 557--567 Kuemmerle, N.B. and Masker, W.E. (1977) J. Virol. 23, 509--516 Masker, W.E., Kuemmerie, N.B. and Allison, D.P. (1978) J. Virol. 27, 149--163 Masker, W.E. and Kuemmerle, N.B. (1980) J. Vixol. 33, 330--339 Seeberg, E., Nissen-Meyer, J. and Strike, P. (1976) Nature 263, 524--525 Masker, W.E. (1977) J. Bacteriol. 129, 1 4 1 5 - - 1 4 2 3 Hanawalt, P.C., Cooper, P.K., Ganesan, A.K. and Smith, C.A. (1979) Ann. Rev. Biochem. 48, 783-836 Carrier, W.L. and Setlow, R.B. (1970) J. Bacteriol. 102, 178--186 Richardson, C.C, (1966) J. Mol. Biol. 15, 49--61 Tsujimoto, Y. and Ogawa, H. (1977) Mol. Gen. Genet. 150, 221--223 McDonell, M.W., Simon, M.N. and Studier, F.W. (1977) J. Mol. Biol. 110, 119--146 Thu~_ng, R.W.J., Sanders, J.P.M. and Borst, P. (1975) Anal. Bioehem. 66, 213--220 Fujimuxa, R.K. (1971) Biochemistry 10, 4 3 8 1 - - 4 3 8 6 Roeder, G.S. and Sadowski, P.D. (1979) Cold Spring Harbor Syrup. Quant. Biol. 43, 1 0 2 3 - - 1 0 3 2 Dressier, D., Wolfson, J. and Magazin, M. (1972) Proe. Natl. Acad. Sci. U.S.A. 69, 9 9 8 - - 1 0 0 2 Rupp, W.D. and Howard-Flanders, P. (1968) J. Mol. Biol. 31, 291--304 Rupp, W.D., Wilde, C.E., III, Reno, D.L. and Howaxd-Flanders, P. (1971) J. Mol. Biol. 61, 25--44 Luria, S.E. (1947) Proc. Natl. Acad. Sci. U.S.A. 33, 253--264 Burek, K.B. and Miller, R.C., Jr. (1978) Proc. Natl. Aead. Sei. U.S.A. 75, 6 1 4 4 - - 6 1 4 8 Burck, K.B., Taylor, D.M., Smith, H.W. and Miller, R.C., Jr. (1979) Cold Spring Harbor Syrup. Quant. Biol. 43, 461--467 Bu~cck, K.B., Scraba, D.G. and Miller, R.C., Jr. (1979) J. Virol. 32, 606--613 Richardson, C.C., Engler, M.J., Kolodner, R.~ LeClerc, J.E., Richardson, D., R oma no, L.J. and Tamanoi, F. (1979) Cold Spring Harbor Syrup. Quant. Biol. 43, 427--440