The arrangement of DNA in lambda phage heads

J. Mol. Biol. (1971) 62, 503-511

The Arrangement of DNA in Lambda Phage Heads II. a DNA after Exposure to Micrococcal Nuclease at the Site of Head-Tail Joining FRANCES

Department

D. GILLINt

AND VERNON

C. BODES

of Biochemistry, University of Maryland Baltimore, Md 21228, U.S.A.

(Received 1 September 19T0, and in revised form

Medical

School

14 July 1971)

DNA in lambda phage heads (lacking tails) is damaged by treatment with micrococcal nuclease. Alkaline sucrose gradient sedimentation of the DNA extracted from nuclease-treated and untreated heads reveals no hydrolysis of internal phosphodiester bonds. Transformation of helper-infected cells by whole or half molecules of treated DNA is as efficient as by control DNA. Thus, the site of nuclease damage must be near the ends and very limited. Below 2O”C, DNA from nuolease-treated heads forms circles in vitro more rapidly than control DNA, presumably by hydrogen bonding at the singlestranded ends. In the control DNA, these bonds melt at 61°C, but in the treated DNA they melt at 36°C. The data suggest that X DNA molecules are positioned in phage heads so that a few terminal bases of the single-stranded 5’ ends are exposed to micrococcal nuclease when it attacks at the site for tail attachment.

1. Introduction paper presents the rationale for this investigation and shows that, if altered by micrococcal nuclease treatment (Bode & Gillin, 1971). The DNA is sensitive to the nuclease only while the head lacks a tail. Therefore, the attack is presumed to occur just outside or through a small opening at the tail-attachment site. Several other n&eases are unable to catalyze head inactivation due either to their specificity or to their size. Micrococcal nuclease was selected for this study because it is small and because it exhibits a broad substrate specificity. The enzyme prepared from the Foggi strain of Staphylococcus aureus is a well-studied protein of molecular weight 17,000 (Heins, The

preceding

a h head is tailless, the DNA inside is functionally

Suriano,

Taniuchi

& Anfinsen,

1967). Its hydrodynamic

properties

are those

predicted

globular molecule of ellipsoidal shape with axes of 2 nm and 8 nm. Although its initial rate of attack is five- to tenfold greater on denatured DNA than on native DNA, large amounts of micrococcal nuclease hydrolyze native DNA to the level of mono- and dinucleotides. It prefers to cleave as an endonuclease at internal dNp--dAp and dNp-dTp bonds but this specificity is not absolute. It also degrades as an exonuclease from the 3’ terminus, yielding 3’ mononucleotides (Cuatrecasas, Fuchs & for a

t Present address: Laboratory of Biochemical Genetics, National National Institutes of Health, Bethesda, Md. 20014, U.S.A. $ Present address: Division of Biology, Kansas State University, Please send reprint requests to this address. 503

Heart

and Lung Institute,

Manhattan,

Kansas

66502.

504

Anfinsen, substrate

F.

1967; Laskowski, for this enzyme.

D.

GILLIN

AND

V.

C. BODE

1967). Thus, almost any exposed DNA might be a

2. Materials and Methods Solutions, procedures and terminology with the additions listed below.

are exactly

(a) Isolation

as described

in Bode

$ Cillin

(1971)

of DNA

DNA was prepared from purified MN-treated? or control hsusJ27 heads by phenol extraction as described by Kaiser & Hogness (1960). Recrystallized sodium dodecyl sulfate, 0*0750/O in Tris-EDTA buffer was included in the extraction mixture to ensure inactivation of micrococcal nuclease. Without dodecyl sulfate, a slight contamination by nuolease was observed. Extracted DNA was dialyzed against Tris-EDTA buffer and stored at 4°C under sterile conditions. (b) Irlfectivity The infectivity (1965) and that

of h DNA whole of h half-molecules

assay for X DNA

molecules was measured as described by Kaiser

(c) Shearing

of h DNA

as described by Bode & Kaiser & Inman (1965).

to half-molecules

X DNA was sheared following the procedure of Kaiser & Inman (1965). Solutions containing less than 10 pg DNA/ml. in Tris-EDTA were sheared at 1200 rev./min for 30 min at 0°C with a single Virtis homogenizer blade (3.9 cm long) in a diagonally fluted V&is flask. Transformation assays revealed that less than 0.5% of the DNA remained as whole molecules. The r\ immunity gene was used as a measure of the right half of the DNA molecule and SUSA +B+ as a measure of the activity of the left half of the molecule.

3. Results (a) Sedimentation

of MN-DNA

at alkaline

pId

Because h heads are somewhat unstable (Harrison & Bode, 1969) and DNA from degraded heads is present in our preparations, we did not attempt to follow the release of 3H-labeled nucleotides into acid-soluble material during MN treatment. Breakdown of O*Ol% of the heads with subsequent hydrolysis of their DNA would mask MN action on DNA in intact heads. Instead, to determine how extensively MN degrades the DNA inside heads, 3H-labeled DNA extracted from MN-treat,ed and control heads was sedimented on alkaline sucrose gradients. The radioactivity profiles failed to reveal a decrease in the single-strand molecular weight of DNA extracted from the inactivated heads (Fig. 1). Thus, if the reduced infectivity observed after head-tail joining (Bode & Gillin, 1971) is due to n&ease action on DNA, degradation must be of limited extent and occur exclusively near the ends of the molecule. (b) Biological activity of MN-DNA Normally the 5’ end of each DNA strand in mature X DNA extends past the 3’ terminus of the other strand for a distance of 12 bases (Wu & Kaiser, 1968; Wu & Taylor, 1971). The structure of these ends is shown in Figure 2. In order to be biologically active in the helper-infected cell transformation system, X DNA molecules or fragments thereof, must possess at least one free single-stranded end (Kaiser & Inman, 1965). DNA extracted from MN-treated and control heads was used in the t Abbreviation

used: MN, micrococcal

nucloase.

DNA

DEGRADED

AT

THE

SITE

OF

HEAD-TAIL

JOINING

605

Fraction number

FICX 1. &us J27 heads were incubated at 37°C for 30 min either with 10 units of MN or with enzyme diluent alone. The heads were repurified to remove any unpackaged DNA by sedimentation through a 15 to 35% sucrose gradient. Those fractions which contained significant numbers of heads were pooled and the DNA extracted with phenol. After dialysis, l-ml. samples were denatured by the addition of 0.1 ml. of 1 N-NaOH and 0.3 ml. of a s2P-labeled X DNA marker was added. Samples (1.0 ml.) were layered on 36-ml alkaline sucrose gradients (0.7 M-NaCl, 0.3 M-N&OH, 10M2 M-TrisHCl, 10e3 M-EDTA with sucrose concentration varying from 5 to 25%) and sedimented for 15.5 hr at 25,000 rev./mm., in a Spinco SW27 rotor at 5°C. Thirty equal fractions were collected, the DNA precipitated with 1.5 vol. of 10% triohloroaoetic acid, the precipitate collected on glass fiber filters and the sH disintegrations counted (Bode & Kaiser, 1965). For comparison, the two gradients are superimposed using the e2P-marker DNA zones control. (not shown) as a reference. - l - 0 -, Treated; --- x --- x ---,

“. The i’ activities of whole DNA molecules transformation of the X immunity gene, % isolated from control and MN-treated phage heads were identical (Table 1). The experiments in the Table were performed near the upper limits of linearity for the assay. In other experiments, where the transformation efliciency was lower, the DNA concentration of whole molecules was varied between 5 x 10V2 and 5 x 10m4 pg/ml. The assay is linear over this range and the efficiencies for MN and control DNA differ by less than 10%. Thus at least one end is sufficiently intact to render the DNA molecule infectious. To determine whether both ends of the MN-DNA are individually active in the transformation assay, DNA molecules were broken by shearing and the resulting half molecules were assayed for their ability to transfer SW A+B + or i” (Kaiser C%

3’ ~5’GGGCGGCGACCT Left end

SUSAB

II II

/

ix

I I I

I

CCCGCCGCTGGA5’ Right end

FIG. 2. Base sequence of the oohesive ends of the X DNA molecule (Wu & Taylor, 1971). In this representation of the X DNA molecule, the dotted line indicates the approximate site where the whole molecule is sheared to halves. The ends are enlarged out of proportion to the remainder of the Figure. 33

F. D. GILLIN

506

AND TABLE

V. C. BODE 1

Biological activity of DNA extracted from MN-treated heads Transformation ia

MN-DNA Control DNA

MN-DNA Control

DNA

Whole A DNA moleoules 12 13 Half molecules of X DNA 4.5 4.0

activity sus A+B+

6.3 6.5 1.5 1.4

Helper-infected cells (0.2 ml. of W3104 pm+ infected with 21hy phage) and DNA (0.1 ml. of solutions containing 0.25 pg/ml.) were mixed at 0°C. DNA uptake took place at 3YC for 20 min before pancreatic DNase was added. Appropriate dilutions were plated on various indicator lawns to detect cells producing phage with genes ia, sus A+B+ or both ia and sus A+B+. The values listed as transformation activity should be multiplied by lo* to obtain the number of cells producing phage with the indicated gene due to 50 pg of DNA. Half molecules were produced by shearing the DNA at 1200 rev./min for 30 mm. For both control and MN-DNA the shearing treatment reduced the occurrence of linked &us AB activity to less than 0.5% the initial value indicating the molecules were in faot broken. The in gene is on the right half of the XDNA molecule while genes

A and B are on the left half.

Inman, 1965; Radding & Kaiser, 1963). Cistrons A and B are located on the left extremity of the molecule and their activity reflects the functional integrity of the left end. Similarly, the i” gene is located on the right half of the molecule and serves as a measure of integrity of the right end (see Fig. 2). The transformation efficiencies for these genes obtained with MN-DNA halves do not differ significantly from the respective efficiencies for control DNA halves (Table 1). The lower absolute efficiency for the genes associated with the left half is a poorly understood characteristic of the assay system and has been observed by other workers (Radding & Kaiser, 1963). In this experiment, the i” activity of the right half was also somewhat lower than that of the whole molecule. The transformation assay was repeated at 30°C using the same preparation. For both MN and control DNA halves the ia efliciency increase by a factor of 1.5 but the SW A+B+ efficiencies decreased by a factor of 2. With two preparations examined under several conditions, the MN and control halves always had equal activity. Thus, the DNA molecules from MN-treated heads retain both single-stranded end sequences sufficiently intact to satisfy the requirements of the transformation assay. (c) Formation and melting of hydrogen-bonded circles The ability of h DNA molecules to form circles in vitro depends upon hydrogen bonding between the two complementary single-stranded regions (Fig. 2). When MN and control DNA preparations are incubated in TCM buffer at temperatures between 5 and 2O”C, MN-DNA not only circularizes, but does so more rapidly than does control DNA (Fig. 3). These results indicate that the ends of the molecules are modified by MN but that the number of bases removed from the 5’ termini is too small to prevent circle formation. The loss of several bases from the 5’ ends should result in a decrease in the temperature, X,, at which 50% of the non-infectious hydrogen-bonded circles open to form

DNA

DEGRADED

AT

THE

SITE

OF HEAD-TAIL

JOINING

507

80

0

I

I

I

I

6

12

18

24

Time (hr) FIG. 3. The rate of hydrogen-bonded circle formation for MN and control X DNA at various temperatures. MN and control DNA preparations were diluted in TCM buffer to 0.25 pg/ml. and incubated at the indicated temperatures. At various times, samples were withdrawn for the transformation assay in which only linear molecules are active. To determine the total number of h DNA molecules and correct for any variations in recipient cells, a sample was withdrawn 10 min prior to each transformation and heated to 76°C for 10 min, then chilled rapidly and assayed immediately along with the unheated sample. The number of plaques observed with the unheated sample times 100 divided by the number observed with the heated sample is plotted as the peroentage of linear molecules. ), MN-DNA; (----), control. F-----

infectious linear molecules. As is shown in Figure 4, the melting temperature, T,, of control DNA circles in TCM buffer is 61°C while that of MN-DNA circles is 36°C. The assay for MN-DNA linear molecules in this and the previous experiment (Fig. 3), might be influenced by circle closing and opening during the transformation assay. Nevertheless, very little difference was noted between assays performed at 30°C and at 37°C. Since the specific biological activity of MN-DNA determined by assay at 37°C is the same as control DNA, either the formation of circular molecules during assay is not a serious problem or MN-DNA is inherently more active. The pattern of results obtained clearly show the data are at least qualitatively significant and, for the reasons indicated above, we believe 36°C is a valid T, for MN-DNA. That the loss and reappearance of transforming activity are actually correlated with circle formation and opening respectively, was confirmed by sedimentation studies, After cyclization, but before heating, 71% of the 3H-labeled MN-DNA and 75% of the control DNA sediments at a rate 1.15 times faster than linear molecules, characteristic of hydrogen-bonded circles, Figure 5. After heating at 42”C, only 25% of the MN-DNA sediments at the rate of circles, but the amount of circular control DNA is essentially unchanged (79%). Heating at 65°C opens at least 90% of the control DNA circles but 34% of the MN-DNA again sediments faster than linear molecules. Since even without heating all of the 3H-labeled MN-DNA co-sediments with the linear 3?P marker if the sedimentation buffer contains only TrisEDTA rather than TCM buffer or Tris-EDTA plus 1 M-Nacl, the circular MN-DNA

508

F.

D.

GILLIN

AND

xI_________ x----. x-x----x-I I L 25 35 45

V.

;5

C. BODE

I

/

65

75

Temperature PC)

FIG. 4. Melting curves for MN-DNA and control DNA hydrogen-bonded circles. MN-DNA and control DNA samples were diluted to 0.2 pg/ m 1. in TCM buffer, heated to 76°C and allowed to cool very slowly overnight to form hydrogen-bonded circles (Wu & Kaiser, 1968). Samples of the DNA were heated for 10 min at the temperatures shown, then chilled rapidly in ice water. Transormation assays were performed to determine the number of linear molecules present. The maximum transformation activity obtained was taken to be 100°/e linear molecules. This may be an underestimate in the case of MN-DNA due to recyclization both before and during assay. Incubating MN-DNA circles for longer than 10 min at 36°C did not change the fraction of linear molecule &s measured in the subsequent trsnsformation assay. - l - l -, MN-DNA; -- x -- x --, oontrol DNA.

molecules can be quantitatively converted to linear ones. That which appears in the gradients carried out after heating to 42°C or to 65°C (Fig. 5) is attributed to a rapid recyclization of MN-DNA during the rather long sedimentation. Although the difference between the rates of circle formation and opening for MN-DNA and for control DNA may complicate the quantitation of circular molecules by the biological assay as well as by the sedimentation assay, both show that MN-DNA circles melt below 42°C indicating that cohesive ends are altered.

4. Discussion The properties of MN-DNA suggest that its alteration involves the loss of only a few bases from its cohesive ends. Since the molecular weight of MN-DNA singlestrands is not detectably reduced (Fig. l), the damage does not involve the hydrolysis of bonds in the central 80% of the molecule. Since MN-DNA forms hydrogen-bonded circles (l?igs 3 and 5) and, after breakage by shear, both halves are active in transformation (Table l), it must still retain at least a portion of both 5’ single-stranded ends. Since the circle melting curve (Fig. 4) is sharp and complete, a similar number

FIG. 5. Sedimentation of circular MN-DNA and control DNA after heating. Tubes containing O-4 pg 3H-labeled DNA/ml., circularized as described in Fig. 4, were heated for 10 min at 42°C or 65°C and cooled rapidly in ice-water. 3aP-labeled h DNA, 2 pg/ml., was added as s marker and 1.0 ml. of each solution layered on a 35 ml. gradient of 5 to 25% sucrose in Tris-EDTA with 1 M-NaCI added. Sedimentation proceeded for 15 br at 25,000 rev./min in the Spinco SW27 rotor. Samples (1 ml.) were collected from the bottom of the tube and the DNA precipitated and counted. Tritum recoveries varied between 87 and 125%. -O--O-, 3H; --X--X--, aaP.

DNA

DEGRADED

AT THE

SITE

OF HEAD-TAIL

JOINING

3oc 2oc IOC c 300

?\

42k

,

2oc IOC C 65°C

300 200 100 z E 3 2

0 .

Control DNA

300

5’C

200 100 0 42T

,300 200 100 0 3oc 2oc IOC C

5 cus Fraction number

FIG. 5.

609

510

F.

D,

GILLIN

AND

V.

C. BODE

of bases probably is removed from every molecule, If the number varied, a broadened melting range would be expected. We will estimate the number by comparing our data for MN-DNA with that obtained in other studies of h DNA with altered cohesive ends. When exonuclease III removed an average of 20 bases from the 3’-OH ends of h (Wang & Davidson, 1968), the circle to linear melting temperature decreased from a value of 62°C to one of 38°C and did not drop further even when the average number lost per molecule was 60 bases. After limited exonuclease III digestion, the cyclization rate of h DNA remained unchanged. Although the Tm of MN-DNA is lowered similarly, to a value of 36”C, its cyclization rate is about ten times faster than control DNA. Thus, the MN and exonuclease III modifications clearly differ. Probably, MN degrades the 5’ not the 3’-OH end of a strand. It is unlikely that a nuclease with the specificity of MN would remove as many as 20 bases from the 3’-OH end without cleaving a single bond in the complementary strand causing the loss of a complete 5’ cohesive end. In another study, Wu & Kaiser (1968) report that the DNA polymerase-catalyzed addition of three bases (all G) to the right 3’-OH end of h DNA reduces the transforming activity of right half-molecules by 25%. (The addition of eight bases, C and G, abolishes it.) Although it altered the biological activity only slightly, the addition of three dG residues lowered the T, of h circles from a value of 62°C to one of 37°C. The addition of three bases to the 3’ end yields a circular molecule with a threebase single-stranded tail; an MN-catalyzed removal of three bases from the 5’ end would yield a circular molecule with a three-base single-stranded gap. In both cases a maximum of nine out of the original 12 bases in a cohesive end could hydrogen bond to stabilize the circular form. The changes in physical and biological properties of h DNA resulting from controlled polymerase or exonuclease III treatment, and especially the properties of h DNA with the right-hand cohesive end shortened due to extension of the duplex region by three base pairs, suggest that the removal of three or four bases from a normal 5’ single-stranded end would yield a molecule with the properties of MN-DNA. The bases that are missing in MN-DNA could be from both single-stranded ends, from one random end, or from one unique end of the DNA molecule. Initial biological experiments indicate they are removed from one unique end and we are verifying this by sequencing. The authors acknowledge the expert assistance given by Sarah Brown and Mr John Blotzer. We are extremely grateful gifts of purified heads and tails and to Dr John Little for help was supported by grants from the National Science Foundation and the Public Health Service (AI-06493 and GM 18182).

Mrs Christina to Mr Dennis

Wong, Harrison

Mrs for

in shearing DNA. This work (GB-6961

and GB 25153)

REFERENCES Bode, V. C. & Gillin, F. D. (1971). J. Mol. Biol. 62, 493. Bode, V. C. & Kaiser, A. D. (1965). J. Mol. Biol. 14, 399. Cuatreoasas, P., Fuchs, S. & Anfken, C. B. (1967). J. Biol. Chem. 242, 1541. Harrison, D. & Bode, V. C. (1969). Genetics, 61, S6. Heins, J. N., Suriano, J. R., Taniuchi, H. & Anfinsen, C. B. (1967). J. BioZ. Chem. 242, 1016. Kaiser, A. D. & Hogness, D. (1960). J. Mol. Biol. 2, 392. Kaiser, A. D. & Inman, R. B. (1965). J. Mol. BioZ. 13, 78.

DNA Laskowski, Radding, Wang, J. Wu, R. & Wu, R. &

DEGRADED

AT THE

SITE

OF HEAD-TAIL

M., Sr. (1967). Achanc. Enzyrnol. 29, 165. C. M. & Kaiser, A. D. (1963). J. Mol. Biol. 7, 225. C. & Davidson, N. (1968). Gold Sp. Harb. Syrnp. Quant. Kaiser, A. D. (1968). J. Mol. Biol. 35, 523. Taylor, E. (19’71). J. Mol. Bid. 57, 491.

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