Terminal repetition in the DNA of bacteriophage T5

J. Mol. Biol. (1972) 69, 187-200

Terminal

Repetition MARO

in the DNA

RHOADES

AND

ELLEN

of Bacteriophage A.

RHOADES

Department of Biology, The Johns Hopkins Baltimore, Md 21218, U.S.A. (Received 19 Januay

T5

University

1972)

The DNA of bacteriophage T5 can be shown to be terminally repetitious by annealing after partial digestion with X exonuclease.Under these conditions circular molecules are formed both before and after repair of the natural singlechain interruptions with polynucleotide ligase. Repaired molecules, however, require less digestion with h exonuclease and yield a higher frequency of circles than unrepaired DNA. These differences,which are not due to digestion at the internal sites in unrepaired DNA, suggest that a single-chain interruption is located within the terminal repetition. This hypothesis wae confirmed by an electron microscopicexamination of circular molecules prepared after extensive digestion with h exonucleaae. These molecules contain short, internal duplex

segmentswhich represent the complementary chains which annealed during circle formation. In repaired DNA the duplex segmentsare of uniform length and give an estimate of 10,000 base pairs (9% of the genome) for the size of the terminal repetition. The duplex segments in unrepaired circular molecules, however, s;re heterogeneous in length and, in most cases, shorter than the terminal repetition. A single-chain interruption can thus be located at variable positions within the repetition at one end of T5 DNA. The repetition at the other end of the molecule does not appear to contain an interruption.

1. Introduction The DNA of bacteriophage T5 possesses several properties which distinguish it from other coliphage DNA molecules.These properties include the presenceof 4 to 5 singlechain interruptions at specific sites within a non-permuted linear genome (Abelson & Thomas, 1966) and the asymmetric breakage of the linear molecule by shear (Burgi, Hershey & Ingraham, 1966; Rubenstein, 1968). In addition, during infection T5 DNA is transferred in a two-step processin which only 8% of the genome is injected in the absenceof protein synthesis (Lanni, 1968). The presence of specific single-chain breaks in T5 DNA was first recognized by Abelson & Thomas (1966), who analyzed the sedimentation profiles of T5 DNA in alkaline sucrose gradients. They proposed a model for the DNA of T&t(O) (a heatstable deletion mutant) in which one chain contains three breaks while the complementary strand has only a single break. Jacquemin-Sablon & Richardson (1970) demonstrated that all of the breaks in the DNA of T5st(O) and T&t(+) (wild type) can be repaired by DNA ligase,indicating that a single phosphodiester bond is missing at each site. These studies also revealed that all of the interruptions in one molecule are confined to one of the two complementary single chains. Bujard (1969) has determined the position of three interruptions in T5st( +) DNA by visualization of partially denatured moleculesin the electron microscope. Two of the breaks are found 187

188

M.

RHOADES

AND

E.

A.

RHOADES

within 20% of the total length from one end of the molecule, while the third is located at 40% from the opposite end. Bujard has proposed that the centrally located break is the site of asymmetric shear breakage and that one of the terminally located breaks separates the first-step transfer DNA from the rest of the molecule. The purpose of the present investigation has been to determine whether the nucleotide sequence in T5 DNA is terminally repetitious. All linear bacteriophage genomes which have been tested have been found to possess either a double-stranded terminal repetition or complementary single-stranded terminals (Thomas $ MacHattie, 1967;

Thomas, Kelly & Rhoades, 1968). Linear bacteriophage genomeswhich contain singlechain interruptions, however, have not been included in these studies. Information concerning the sequence of nucleotides at the ends of T5 DNA is also of interest in

view of the essential role played by one end of the genome in DNA transfer (Lanni, 1969). The experiments reported here demonstrate that the DNA molecules of T5st( +) and T&t(O) are terminally repetitious. In both casesthe repeated region contains 10,000 basepairs, or 9% of a full genome.The two ends of each molecule, however, are not physically identical. A single-chain break is located within the repetition at one end of the molecule, but not at the other. The position of this interruption appears to be highly variable within the terminal repetition.

2. Materials and Methods (a) Preparation of bacteriophage DNA Wild-type bacteriophage T5 and the heat-stable mutant T5st(O) were obtained from the stocks used by Abelson & Thomas (1966). Phage were grown on Escherichia co.% B23 by confluent lysis using the medium described by Rhoades, MacHattie & Thomas (1968) modified to contain 0.05% Casamino acids and 1.2% agar. Radioactively labeled phage were prepared by addition of 32P04 or [3H]thymidine (6.7 Ci/m-mole) at 10 &i/ml. to the bottom agar. The plates were seeded with 5 x lo* bacteria and 1 x lo6 phage particles and incubated for 6 hr at 37°C. Phage were released from the agar in suspension medium (Weigle, Meselson & Paigen, 1959) and purified by differential centrifugation and sedimentation in CsCl step-gradients (Abelson & Thomas, 1966). DNA was isolated by extraction with phenol as described by Rhoades, MacHattie & Thomas (1968). Phenol was removed by extensive dialysis against 0.01 M-TrisHCl (pH 7.9), 1 x 10e4 M-EDTA. Yields of 6 x 1O1l phage-equivalents of DNA/Petri plate (10 cm diameter) were routinely obtained.

The intracellular, replicative forms of #X174,- DNA were prepared according to the procedure of Dressler & Denhardt (1968) with the following exceptions: infection was not synchronized; ohloramphenicol (40 pg/ml.) was added to prevent formation of singlestranded DNA; KCN and iodoacetate were omitted from the lysis buffer. (b) Treatment of T5 DNA with DNA ligaae DNA ligase was obtained from E. coli 1100 (Durwald & Hoffman-Berling, 1968) infected with bacteriophage T4D. Enzyme assays and purification were performed as described by Weiss, Jacquemin-Sablon, Live, Fareed & Richardson (1968). The final product was free of detectable endonucleolytic activity. Reaction mixtures contained T5 DNA at 10 pg/ml., 67 mu-Tris*HCl (pH 7*6), 6.7 mm-MgClz, 1 mM-2-mercaptoethanol, 0.067 mm-ATP, and 0.06 units/ml. of T4 ligase (assayed by ATP-PPI exchange). Incubation was at 37°C for 10 min. The reaction w&s stopped by a single extraction with phenol, followed by dialysis as described above. The product was analyzed by sedimentation in alkaline sucrose gradients. A exonuolease Radding (1966).

(c) Digerrtion of T5 DNA with X exonuclease waa isolated from E. co&i 1100 (XTzl) according to the procedure of Chromatography on phosphocellulose yielded a single peak of activity

TERMINAL

REPETITION

IN

T5

DNA

189

corresponding to free exonuclease (Radding, Rosenzweig, Richards & Cassuto, 1971). Reaction mixtures contained 67 mba-glycine/KOH (pH 9*6), 3 mM-MgCl,, T5 DNA at 2 pg/ml., and fourfold excess of h exonuclease (see text). All incubations were carried out at 22°C. The reaction was stopped by adding 0.1 vol. of 20 xSSC (SSC is 0.15 M-N&I, 0.015 ~-sodium citrate) and chilling to 0°C. The extent of hydrolysis was measured by the production of acid-soluble nucleotides. 0.1 ml. of reaction mixture was mixed with 0.1 ml. salmon sperm DNA, 2.5 mg/ml., and 0.2 ml. 5% trichloroaoetic acid and the resulting precipitate cured at 0°C for 10 min. After centrifugation for 5 min at 5000 g the supernatant fraction was assayed for radioactivity. (d) Chromatography of exonucleme-treated T5 DNA on hydroxyapatite T5 DNA was treated with h exonuclease as described above except that the reaction was stopped by addition of 0.1 vol. of 1-O M-N&~ and heating at 65°C for 5 min. After dilution with 2 vol of 0.01 M-sodium phosphate (pH 6+?), the partially digested DNA was loaded on columns of hydroxyapatite prepared according to the procedure of Tiselius, Hjerten BE Levin (1966). Single-stranded and double-stranded DNA molecules were separated by elution with a linear gradient of 0.01 to 0.50 M-sodium phosphate (pH 6.8). Chromatography was carried out at room temperature. (e) Sucrose density gradient sediment&on Convex exponential gradients of 4.5 ml. were generated with 6 and 25% (w/v) sucrose solutions according to the method of Noll (1967). The mixing volume was 4.5 ml. Neutral sucrose gradients contained 0.01 M-TriseHCl (pH 7.9), 0.15 M-NaCl, 1 x 10m4 M-EDTA, and O.O6o/o sodium dodecyl sulphate. Alkaline gradients contained 0.15 M-NaOH, 0% MNaCl. Samples to be run on alkaline gradients were denatured in 0.1 M-NaOH. Volumes of O-15 ml. or less (containing DNA at 1 to 2 pg/ml.) were added to the top of each gradient with a wide-bore plastic pipette. Centrifugation occurred at 20°C in the SW60.1 ewinging bucket, rotor in a Spinco model L ultracentrifuge. Fractions were collected from the bottom of the tube onto squares of Whatman no. 5 filter paper, dried, and counted in a liquid scintillation counter. (f) Electron microscopy DNA was prepared for microscopy by the protein film procedure of Kleinschmidt & Zahn (1959) aa modified for use with formamide (Westmoreland, Szybalski & Ris, 1969). The final formamide concentration in the spreading solution (hyperphase) was 45% (v/v). The circular, double-stranded replicative-form DNA of bacteriophage +X was used as an internal molecular weight standard. Grids were shadowed with platinum and examined in a RCA EMU-3 electron microscope.

3. Results (a) Chamcterimtion of T5 DNA in alkaline sucrosegradients !l’he single polynucleotide chains derived from T5 DNA sediment in several zones in alkaline sucrose gradients (Abelson & Thomas, 1966). Figure 1 showsthe alkaline gradient profiles obtained for the two DNA molecules used in this study, T5st( +) and T&t(O). Although the bulk of each DNA sediments in three major peaks, a number of minor speciesmust also be present. It will be shown below that the shoulder on peak III

(fractions

28-29)

represents

a distinct

class of single

chains.

The relative

heights

and positions of the peaks in Figure 1 are nearly identical with those obtained by Jacquemin-Sablon & Richardson (1970) for denatured TBst( +) and T&t(O) DNA in high-salt neutral sucrosegradients. (b) Repair of T5 DNA with DNA Zigme The single-chain breaks in T5 DNA can be repaired by the action of T4 DNA ligase (Jacquemin-Sablon & Richardson, 1970). Figure 2 compares the alkaline sedimenta-

190

IM.

RHOADES

AND

Fraction

E.

A.

RHOADES

no.

FIG. 1. Zone sedimentation of T6st( +) and T5st(O) DNA in an alkaline sucrose gradient. saP(-) were mixed, denatured in lebeled TSst( +) DNA (----) and sH-lebeled T6st(O) DNA O-1 M-N&OH, and centrifuged for 106 mm et 40,000 rev./min in an alkaline snorose gradient as described in Materials end Methods. The profiles have been plotted as straight lines connecting the points measnred for each fraction. The direction of sedimentation is from right to left.

tion profiles of untreated and ligase-treated T&t(O) DNA. Most of the ligase-treated DNA co-sediments exactly with the longest chain in the untreated DNA. The absence of slower-sedimenting peaks after ligase treatment indicates that all of the interruptions can be repaired. In agreement with Jacquemin-Sablon & Richardson (1970) this result demonstrates that one chain in mature T6 DNA is continuous over the full length of the duplex molecule. (c) Digestion of T5 DNA

with h exonuclease

Terminal repetition in a linear genome can be revealed by annealing the DNA after limited digestion of both ends by a strand-specific exonuclease. If the DNA is repetitious, the exonuclease will expose complementary nucleotide sequences and the two ends will anneal to form a circular molecule (MacHattie, Ritchie, Thomas & Richard-

Fraction

no

Fro. 2. Zone sedimentation of TW(0) DNA in 8n alkaline sucrose gradient before and after repair with DNA ligase. 3aP-18beled TW(0) DNA (----) was treated with ligase as described inMatsrials and Methods. Untreated sH-labeled T&t(O) DNA ( -) was added and after denaturation in 0.1 M-NaOH, the mixture was centrifuged for 90 min at 40,000 rev./min.

TERMINAL

REPETITION

IN

T5

DNA

191

son, 1967). If the terminal sequences are not complementary, the linear structure will be preserved (Ritchie, Thomas, MacHattie & Wensink, 1967). Previous studies of terminal repetition have utilized exonuclease III of E. coli, which releases 5’-mononuoleotides from the 3’-ends of strands in a DNA duplex. Since exonuclease III can initiate degradation at single-chain interruptions (Masamune, Fleischman & Richardson, 1971), it is not suitable for use with T5 DNA. The exonuclease induced by bacteriophage h, however, has properties similar to those of exonuclease III yet is not active at single-chain breaks (Carter Q Radding, 1971; Masamune et al., 1971). This enzyme is specific for double-stranded DNA, releasing 5-mononucleotides from 5’-ends of polynucleotide chains (Little, 1967). The mode of attack of h exonuclease is processive, or non-random. Each enzyme molecule remains attached to one end of a DNA molecule throughout the course of digestion (Carter & Radding, 1971). In order to obtain uniform degradation of all molecular ends, T5 DNA was digested with X exonuclease in the presence of excess enzyme. Suitable conditions were determined by titrating a fixed amount of DNA with increasing amounts of exonuclease. A titration experiment, performed in parallel on repaired and unrepaired T5 DNA, revealed that both kinds of DNA require the same amount of exonuclease for saturation, and both show the same extent of digestion at saturation. The single-chain breaks in T5 DNA thus do not serve as substrates for h exonuclease. The time courses of digestion of repaired and unrepaired T5 DNA with excess X exonuclease are identical with a linear rate of degradation. If the reaction is allowed to proceed to completion, over 45% of the radioactivity of both molecules is rendered acid-soluble. Since X exonuclease does not degrade single-stranded DNA, this indicates that at least 90% of the DNA is susceptible to attack. The effect of partial digestion with X exonuclease on the alkaline sedimentation profile of T5 DNA is shown in Figure 3. Unrepaired TBst(0) DNA, labeled with [3H]thymidine, was digested to 8% with h exonuclease and centrifuged in the presence of untreated 32P-labeled TBst(0) DNA (Fig. 3(a)). Before digestion the sedimentation profiles of both DNA preparations are nearly identical (Fig. 3(b)). Exonuclease treatment appears to cause degradation of single chains in peaks I and II, but little change in the isotope ratio is seen in the region where the low molecular weight chains sediment. If these chains were attacked, it would be expected that digestion sufficient to degrade 8% of the total DNA would cause a dramatic loss of material in slowsedimenting peaks. Thus the 5’-ends of the shorter single chains must be located at internal interruptions. This experiment has been repeated with both T5st(O) and TSst( +) DNA with similar results. (d) Fornthm

of circulizr DNA

Circular molecules were produced by annealing T5 DNA after partial digestion with h exonuclease. DNA taken directly from exonuclease reaction mixtures was annealed for 60 minutes at 65°C and assayed for circles by zone sedimentation in neutral sucrose gradients. Circular molecules sediment 13 to 14% faster than the corresponding linear form (Hershey, Burgi & Ingraham, 1963). Figure 4 shows the sedimentation profiles obtained for repaired 3aP-labeled TSst(0) DNA annealed after 0, 5.5, and 10*4°h digestion with h exonuclease. Annealing after 10% digestion converts T5 DNA to a form which sediments 13% faster than linear DNA (Fig. 6(a)). At 5% digestion a similar, but less complete conversion has occurred (Fig. 4(b)). It is

192

M.

RHOADES

3

AND

E.

15

1” Fraction

A.

RHOADES

20

25

na

Fro. 3. Effect of p&ial digestion with A exonucleaee on the alkaline sedimentation profile of T6 DNA. Unrepaired 3H-labeled T&‘(O) DNA (----) was centrifuged in an alkaline SUC~OBB gradient before (b) and after (e) 8% digestion with h exonucleese. Untreated 3aP-l&eled T&t(O) DNA () was added to each gradient &a 8 marker. Sedimentation was for 90 min at 40,000 rev./min.

likely that some dimeric linear concatemers have been formed at this stage. In the absence of digestion (Fig. 4(a)) or annealing (not shown) the sedimentation profile remains unchanged. Direct evidence for the formation of circular TS DNA under these conditions was obtained by electron microscopy. Examples of two circular T5 DNA molecules are shown in Plates I and II. There is a perfect correlation between the presence of the fast-sedimenting zone seen in Figure 4 and the appearance of circular structures in the electron microscope. The two ends of the T5 DNA molecule thus contain complementary nucleotide sequences. Circular molecules have been prepared from TSst( +) and T&t(O) DNA, both before and after repair of the single-chain breaks with DNA ligase. In all cases, the repaired and unrepaired molecules were processed identically except for exposure to ligase. Results summarizing the dependence of circle formation upon the extent of exonuclease digestion are shown in Figures 5 and 6. The frequency of circular structures was estimated from sedimentation proBea of the type seen in Figure 4. All material sedimenting between 1-l and 1.3 times as fast as linear DNA was classified as circular. Other fast-sedimenting structures, such as linear dimers, which result from intermolecular annealing have been counted as circles in this analysis. The results in Figures 6 and 6 reveal a large difference in the amount of digestion required to oyclize repaired and unrepaired TS DNA. For both TSst( +) and T5et(O), repair of the single-chain breaks allows e%lcient circle formation to occur after 4 to 5%

I’r..4wa I. A rircular molcrule of ligase-repaired TM(l)) DNA formed by annealing after digestion t’o 12% with X exonuclease. The arrows indicate the boundaries of the internal duplex segment, whxch, in this molecule, represents the terminal repetition. The small circular molecules are +X 174 1)x-A.

PLATE II. A circular molecule of unrepaired TM(O) DNA formed hy iluur~;tling aft(v rligvst ion to 12% with h exonuclease. The &rrows indicate the boundaries of the internal duplex srgmc‘nt which, in this molecule, is equal to 357; of the length of the terminal rrpctition. The small rirruktt molecules are 4x174 DNA.

TERMINAL

REPETITION

200

IN

DNA

(a) LlIiz!J :I 100 ;: : : : 1,

100 __

400

T6

__.,

,.' 1'

193

50 ,.

100 l¶=-dbJ :

50

200

5

IO

15

Fraction

20

no.

FIQ. 4. Formation of circular T5 DNA after digestion with h exonuclew and ennealing a~ seen ( -) by eedimentetion in neutral eucroee gradients. 31P-labeled TSst(0) DNA was digestad to (c) O%, (b) 6.5%. (a) 10.4% with X exonuclease and annealed at 65°C for 60 min in 2 x SSC. Untreated 3H-labeled TSet(0) DNA (----) was added and the mixture centrifuged for 90 min et 40,000 rev./min. A total of 45 fractions w&e collected from eech gradient.

(0) 80-

tp

60 -

. ../

40-

60 -(b)

1. -I

20 t I IO

5 % Acid-soluble

I I 15

3zP

FIG. 6. Formation of circular Tti( +) DNA 88 8 function of the extent of digestion with X exonuclease. Samples of sap-labeled (a) repaired and (b) unrepaired T&(O) DNA were digested with A exonucleaae, ennealed, and anelyzed by sedimentation in neutral sucrose gradients. The frequenoy of circuler DNA was determined aa described in the text.

194

M.

RHOADES

AND

E.

A.

RHOADES

60

% Acid-soluMe3’P

Fm. 6. Formation of circular T&t(O) DNA as a function of the extent of digestion with exonuclease. Samples of (a) repaired and (b) unrepaired T&G(O) DNA were treated as described the legend to Fig. 6.

X in

of the nucleotides have been rendered acid-soluble. In both cases over 80% of the DNA is converted to a fast-sedimenting form. In contrast, the unrepaired molecules require about 8% digestion and the frequency of circular molecules does not exceed 50%. The average extent of digestion needed to cyclize each type of DNA can be estimated from the curves in Figures 5 and 6. These values are given in Table 1 along with the number of nuoleotides removed per single chain. The Snding that ligase-treated DNA will cyclize after significantly less exonucleolytic digestion than is required for unrepaired DNA suggests that a single-chain

Extent

DNA

TS&( +)

TFW’) Repaired

TABLE I required for cyclization

O/e Digestion

TM+) Repaired

of digestion

TSet(O)

‘7.8 4.6 8.6 6.6

The percentage digestion required for cyclization points of the curves in Figs 6 and 6. Values of 76.6 weights for the sodium salts of T68t( +) and T&(O) on a ratio of 20-9 for the relative molecular lengths (molecular weight 3.4 x 10e; Sinsheimer, 1959). The T68t( +) DNA (Rubenstein, 1968).

Number of nucleotides removed per chain 8900 6160 9100

6900 was estimated from the position of the midx 10s and 71.0 x 10s were used as molecular DNA, respectively. These values are based of TSat(0) DNA to double-stranded #X DNA molecular weight of T68t(O) DNA is 0.94 of

TERMINAL

REPETITION

IN

TS

DNA

195

break is located within the terminal repetition at one end of the molecule. A model illustrating this situation is shown in Figure 7. The hypothetical interruption has been placed near the middle of the repetition at the 3’-end of one strand. Unless this interruption is repaired by ligase, treatment with X exonuclease will release the short, terminally located polynucleotide as the complementary strand is degraded. Loss of this chain will create a need for additional digestion before complementary sequences are exposed at both ends.

A exonuclease i

FIU. 7. Model for T6 DNA in which a single-chain interruption is located within the terminal repetition. The two complementary chains of the duplex are depicted by parallel lines. The terminel repetition is indicated by the letters A to G. As the 5’-ends are degraded by A exonucleaea the short polynucleotide D’E’F’G’ will be lost from the end containing the interruption. Further digestion will expose the sequences A’B’C’ and allow annealing to occur.

:Further information on this feature of T5 DNA has been obtained by electron microscopy of circular molecules prepared after extensive digestion with X exonuclease. Once exonucleolytic digestion has proceeded past the boundaries between the terminal repetitions and the interior of the DNA, the resulting circular molecules will contain two internal single-stranded regions (Ritchie, Thomas, MacHattie & Wensink, 1967). The duplex DNA lying between the single-chain regions represents the complementary chains which annealed during circle formation. In a molecule where single-chain breaks are absent, the length of the internal duplex segment is equal to the length of the terminal repetition. The presence of a terminal interruption (Fig. 7), however, should result in a circular molecule with an internal duplex segment which is shorter than a full repetition. Short internal segments of double-stranded DNA bracketed by single-stranded regions can be seen in the circular molecules shown in Plates I and II. Histograms of the lengths of the internal duplex segments found in repaired and unrepaired circular T5 DNA are shown in Figure 8. The results for TBst( +) and T5st(O) have been pooled and expressed in units of nucleotide pairs. The internal duplex segments in repaired T5 circles are homogenous in length and provide an estimate of 9500 to 10,000 base pairs for the size of the terminal repetition (Fig. 8(a)). The results in Table 1 indicate that an average of only 5500 nucleotides removed per single chain is sufficient to 14

196

MI.

RHOADES

AND

E.

A.

RHOADES

I (a)

IO-

J Nucleatlde

pairs

FIG. 8. Lengths of internal duplex segments seen in circular TS DNA. Circular molecules of (a) ligase-treated and (b) untreated T5 DNA, prepared by annealing after 10 to 12% digestion with h exonuclease, were examined in the electron microscope. Length measurements were made of the short duplex segments found in all intact circular molecules which contained two singlestrcmded regions. The average length of at least 3 untwisted, circular molecules of 4X DNA on the same micrograph was used to calculate the number of nucleotide pairs in each T5 segment. The DNA, to the nearest 500 nucleotide solid line indicates the pooled results for TSst( + ) and TM(O) pairs. The shaded area represents the histogram obtained for T54 +) DNA alone.

cyclize repaired molecules. Efficient annealing can thus occur before digestion has exposed the complete repetition at both ends of a molecule. This would be expected since complementary sequences will be exposed as soon as the sum of the nucleotides removed from each molecule exceeds the number of nucleotide pairs in the repetition. In contrast, the internal duplex segments in circular molecules prepared from unrepaired T5 DNA are heterogeneous in size (Fig. 8(b)). Some values of 10,090 base pairs were found, but most of the segments contain between 1500 and 7500 base pairs. The majority of these molecules contain a single-chain break within the terminal repetition, but the position of the interruption is highly variable within this region. If the size of the repetition is constant, this suggests that either the interruption is not located at a specific nucleotide sequence, or that the terminal sequence of nucleotides is variable in T5 DNA. (e) Isolation of the terminal single chain The hypothesis presented above predi&s that a class of short single chains should be released from unrepaired T5 DNA after limited digestion with h exonuclease. In order to test this possibility, T5 DNA was degraded to various extents with X exonuclease and then chromatographed on hydroxyapatite to isolate free single chains. The results in Figure 9 show that single chains are released from unrepaired T5 DNA at a nearly constant rate beginning at an early point in the exonuclease reaction. The rate of release of single chains from repaired T5 DNA is considerably lower. Figure 10 shows the alkaline sedimentation pattern obtained for the single-stranded DNA released from unrepaired T5 DNA after 12% digestion. This DNA sediments in a single zone corresponding to the position of the shortest chains which can be produced by denaturation

TERMINAL

REPETITION

IN

T5

197

DNA

I In 15 .E 2 0 +i .E In ‘0 0 24 idti 5 $

/’ /’ *---

./-

/ /’ .---• ./” L&f

.’ 5

___---/-CD---^_ --l IO I5 --h-+--

-0 ?k--

5

% Acid-soluble

Fro. 9. Release of single chains from TS DNA as a function of extent of digestion exonuclease. Samples of eeP.labeled repaired (--O---O--) and unrepaired (-•-~---) T5&( +) DNA were digested with X exonuclease and the reaction mixtures chromatogrsphed hydroxyapatite as described in Materials and Methods. The amount of single-stranded recovered is expressed as a percentage of the total radioactivity applied to each column.

Fraction

with

h

on DNA

no.

Fra. 10. Alkaline sedimentation of the single chains released from T5 DNA by limited digestion with h exonuclease. ssP-labeled T5et( +) DNA (----) was degraded to 12% with X exonuclease and the released single chains collected by chromatography on hydroxyapatite. A portion of the previously purified single-stranded DNA was mixed with eH-labeled T5at( +) DNA ( ----), denatured in 0.1 M-NaOH, and centrifuged for 95 min at 40,000 rev./min in an alkaline sucrose gradient.

of a whole molecule. When digestion w&s allowed to proceed further, DNA sedimenting in the region of peak III (fraction 19) was also recovered from hydroxyapatite. The position of the peak of single-stranded DNA in the gradient in Figure 10 correaponds to fragments of 3 to 5% of an intact T5 strand (Abelson & Thomas, 1966). This is equivalent to 3500 to 6000 nucleotides and is consistent with the presence of a singlechain interruption within the repetition at one end of T5 DNA.

4. Discussion The results presented here demonstrate that the terminal nucleotide sequences in T5 DNA are identical, or very closely related. Previous studies have shown that the DNA molecules of bacteriophages T2, T3 and T7, P22, Pl, coliphage 15 and Tl are

198

M.

RHOADES

AND

E.

A.

RHOADES

terminally repetitious (MacHattic et al., 1967; Ritchie et al., 1967; Rhoades et al., 1968; Ikeda & Tomizawa, 1968; Lee, Davis & Davidson, 1970; MacHattie, Rhoades & Thomas, manuscript in preparation). T5 DNA represents the first instance of a terminally repetitious duplex molecule which contains natural interruptions. The addition of T5 to the above list strengthens the hypothesis (Thomas, 1966,1967)that terminal repetition is a universal feature of linear bacteriophage genomes. The size of the terminal repetition in T5 DNA was estimated from electron micrographs of circular molecules prepared after extensive digestion with h exonuclease. These molecules contain a short, internal duplex segment which, in ligase-treated DNA, represents the terminal repetition. Measurement of these segmentsfor TB.st(+) and T&t(O) DNA gives a value of approximately 10,000 base pairs, or 9% of the genome. The size of the repetition appears to be constant, although small variations cannot be excluded. This is the largest terminal repetition which has been reported to date for a virus with a unique nucleotide sequence. The first-step transfer segment of the T5 genome has been estimated to contain 8 to 8.5% of the DNA (Lanni, 1968). Although the first-step transfer region and the terminal repetition may not be exactly identical, it is clear that most of the sequences which are initially transferred are repeated at the other end of the genome. The firststep transfer region is known to contain at least three genes, two of which supply functions required for the completion of DNA transfer (Lath, 1968; Hendrickson & McCorquodale, 1971). The third gene, identified in the first-step transfer segmentsof both T5 and the related phage BF23, is responsiblefor restriction in host cells carrying colicinogenic factor Ib (Mizobuchi, Anderson & McCorquodale, 1971; McCorquodale, personal communication). The existence at this locus of mutations which are recessiveto wildtype and do not block DNA transfer is difficult to reconcile with the apparent diploid nature of the first-step transfer DNA. Either the repetition distal to the first-step transfer region is genetically inert or the Ib restriction gene must act before completion of DNA transfer. Cyclization of T5 DNA was found to be facilitated by repair of the natural interruptions with polynucleotide ligase. Repaired molecules require less digestion with h exonuclease and yield a higher frequency of circles than unrepaired molecules. These differences are not due to digestion at the internal 5’groups in unrepaired DNA since h exonuclease was found to act identically on both kinds of molecules. The requirement for additional digestion could be explained if a single-chain break is located within the terminal repetition near the 3’-end of one strand. As indicated in Figure 7, digestion with h exonucleasewill result in lossof a short polynucleotide which contains sequencescomplementary to those exposed at the other end of the molecule. Interruptions located outside the terminal repetition, however, would not affect the amount of digestion required for initial cyclization. Evidence supporting this hypothesis was provided by electron micrographs of circular moleculesprepared after extensive digestion with h exonuclease. Unrepaired T5 DNA will cyclize under these conditions, but most of the circles contain internal duplex segmentswhich are shorter than the terminal repetition. This indicates that treatment with h exonuclease results in the removal of nucleotides from one of the external 3’-ends of T5 DNA. Chromatography of a reaction mixture on hydroxyapatite revealed that the missingDNA is releasedin the form of short single chains. Since these chains were shown by sedimentation analysis to represent 3 to 5% of an intact T5 strand, an interruption must be located near the middle of the repetition at one end

TERMINAL

REPETITION

IN

T5

DNA

199

of the molecule. The repetition at the other end of T5 DNA, however, does not appear to contain a single-chain break. This follows from the alkaline sedimentation analysis (Fig. 3) of unrepaired T5 DNA after partial digestion with h exonuclease. Digestion was found to affect only chains in peaks I and II, which represent an intact T5 strand and a fragment of approximately 40% of an intact strand (Abelson & Thomas, 1966). Since X exonuclease attacks only external 5’-termini, an interruption cannot be located within 40% of one end of the T5 duplex. An unexpected tiding was the variation in length of the short duplex segments in unrepaired circular molecules. These segments, which represent the DNA between the beginning of the repetition and the terminal interruption, were found to contain 1500 to 7500 base pairs. Since the repetition contains approximately 10,000 base pairs, a single-chain break can be located anywhere from 2500 to 8500 base pairs from one end of the T5 duplex. This conclusion, however, only applies to that fraction, approximately 50%, of unrepaired DNA which is capable of circle formation after digestion with X exonuclease. In an earlier study, Bujard (1969) observed that 5 to 10% of the molecules in a population of partially denatured TBst( +) DNA contain an interruption at 8% from one end. Since an interruption at this location would result in loss of most or all of the repetition after extensive digestion with h exonuclease, molecules of this type would not have been observed in the present study. An interruption located at 8% from one end, however, would not be expected to influence the amount of digestion required for initial cyclization. As shown in Figures 5 and 6, circular unrepaired molecules cannot be detected at 5% digestion, a point at which repaired DNA cyclizes efficiently. It seems likely that most T5 DNA molecules contain an interruption near the middle of the repetition regardless of whether a second break is located at 8% from one end. The variable location of the terminal interruption in T5 DNA can be interpreted in one of two ways. It is possible that this interruption occurs at variable positions in the nucleotide sequence of T5 DNA. Alternatively, the interruption could occupy a fixed position within a variable nucleotide sequence. T5 DNA, for example, might exist in several terminally repetitious, permuted sequences, all of which start and finish within the same region of the genome. If the terminal repetitions were identical in size, but differed in sequence, the results obtained in this study could be explained. Complete denaturation and reannealing of ligase-treated T5 DNA, however, yields predominantly linear moleculeswith duplex ends (P. Scheible & M. Rhoades, unpublished experiments). Although small variations cannot be excluded, this result suggests that all T5 DNA molecules are identical in both length and nucleotide sequence. We are grateful to Dr Bernard Weissfor a timely gift of polynucleotide ligate and to Dr Evangelos Moudrianakis, Mr David Longfellow and Mr James Diven for assistance with electron microscopy. This work was supported by the National Science Foundation grant no. GB-20460. This ia contribution no. 687 from the Department of Biology, The Johns Hopkins University, Baltimore, Maryland. REFERENCES Abelson, J. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Bujard, H. (1969). Proc. Nut. Acud. Sci., Fada. 62, 1167. Burgi, E., Hershey, A. D. & Ingraham, L. (1966). V&oZogy, 28, 11. Carter, ID. M. & Radding, C. M. (1971). J. Biol. Ch.em. 246, 2502. Dressler, D. H. & Denhardt, D. T. (1968). Nature, 219, 346.

M.

200

RHOADES

AND

E. A. RHOADES

Durwald, H. & Hoffman-Berling, H. (1968). J. Mol. Biol. 34, 331. Hendrickson, H. E. & McCorquodale, D. J. (1971). J. Viral. 7, 612. Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc. Nut. Acd Sci., Wash. 49, 748. Ikeda, H. & Tomizawn, J.-I. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 791. Jacquemin-Sablon, A. & Richardson, C. C. (1970). J. Mol. Bid. 47, 477. Kleinschmidt,, A. K. & Z&n, R. K. (1959). 2. Natwj. lab, 770. Lanni, Y. T. (1968). Bad. Rev. 32, 227. Lanni, Y. T. (1969). J. MOE. BioZ. 44, 173. Lee, C. S., Davis, R. W. & Davidson, N. (1970). J. Mol. BioZ. 48, 1. Little, J. W. (1967). J. BioZ. Chem. 242, 679. MacHattie, L. A., Ritchie, D. A., Thomas, C. A., Jr. & Richardson, C. C. (1967). J. MOE. BioZ.

23, 355.

Masamune, Y., Fleischman, R. A. & Richardson, C. C. (1971). J. BioZ. Chem. 246, 2680. Mizobuchi, K., Anderson, G. C. & McCorquodale, D. J. (1971). Genetics, 68, 323. Noll, H. (1967). Nature, 215, 360. Radding, C. M. (1966). J. Mol. BioZ. 18, 235. Radding, C. M., Rosenzweig, J., Richards, F. & Cassuto, E. (1971). J. BioZ. Chem. 246, 2510. Rhoades, M., MacHettie, L. A. & Thomas, C. A., Jr. (1968). J. Mol. BioZ. 37, 21. Ritchie, D. A., Thomas, C. A., Jr., MacHattie, L. A. & Wensink, P. C. (1967). J. Mol. BioZ.

23, 365.

Rubenstein, Sinsheimer, Thomas, C. Thomas, C. Thomas, C.

I. (1968). Virology, 36, 356. R. L. (1959). J. Mol. BioZ. 1, 43. A., Jr. (1966). Progr. Nucleic Acid Res. Mol. BioZ. 5, 315. A., Jr. (1967). J. Cell. PhyeioZ. 70, Sup. 1, 13. A., Jr., Kelly, T., Jr. & Rhoades, M. (1968). Cold Spr. Harb. Symp. Quunt. BioZ. 33, 417. Thomas, C. A., Jr. & MaeHattie, L. A. (1967). Ann. Rev. B&hem. 36, 485. Tiselius, A., Hjerten, S. & Levin, 0. (1956). Arch. B&hem. Biophys. 65, 132. Weigle, J., Meselson, M. & Paigen, K. (1959). J. Mol. BioZ. 1, 379. Weiss, B., Jacquemin-Sablon, A., Live, T. R., Fareed, G. C. & Richardson, C. C. (1968). J.

BioZ.

Westmoreland,

Chem.

243,

4543.

B. C., Szybalski,

W. & Ris, H. (1969). Science, 163, 1343.