Arrangement of DNA in lambda bacteriophage heads

J. Mol. B&d. (1972) 69,201-207

Arrangement III.?

Location

of DNA

in Lambda Bacteriophage

Heads

and Number of Nucleotides Cleaved from h DNA Micrococcal Nuclease Attack on Heads R. PADMANABHAN,RAY

by

WV

Section of Biochemistry and Molecular Biology Cornell University, Ithaca, N. Y. 14850, U.S. A. AND VERNON C. BODE

Division of Biology, Kansa-sState University Manhattan, Kansas 66502, U.S.A. (Received22 Pebruary 1972, and in revisedform 6 May 1972) If lambda phage heads are exposed to micrococcal nuclease before phage tails are attached, the cohesive ends of the packaged h DNA are damaged. The altered ends were used as templates for repair synthesis catalyzed by Escherichiu coli DNA polymerase I. The set of nucleotides added to the 3’-termini of DNA from micrococcal nuolease treated lambda heads was compared with that added to the DNA from control heads not exposed to this nuclease. The comparison indicates that micrococcal nucleate treatment of heads removes four terminal bases from the right-hand cohesive end of the packaged DNA, but leaves the left-hand cohesive end intact.

1. Introduction DNA in lambda phage particles is not degraded in the presenceof deoxyribonucleases. Presumably the phage coat-proteins shield it from molecules as large as enzymes. During phage morphogenesis,DNA-filled heads are formed by one seriesof reactions and the tail assembly by another, independent, series. The nuclease sensitivity of DNA in h headswas investigated by Bode & Gillin (1971) ; it was found that until the head joins with a tail, packaged DNA is susceptible to a very limited degradation catalyzed by micrococcal nuclease (MN)$ Presumably the degraded portion of the /\ DNA molecule is exposed at the tail attachment site. When the altered DNA extracted from lambda heads after treatment with micrococcal nuclease was examined, it was found that degradation had occurred only at the cohesive ends of the molecule and that only 3 or 4 nucleotides were removed (Gillin & Bode, 1971). The experiments did not establish whether nucleotides were removed from both single-stranded ends of the molecules, from one end selected at random or from

one unique

end. Studies

of the melting

of cohered

7 Paper II in this series is Gillin & Bode, 1971. This paper Nucleotide Sequence Analyaie of DNA. Paper VII is Donelson $ Abbreviations used: MN, micrococcal nuclease; MN-DNA, 201

half-molecules,

is also paper VIII & Wu, 1972. DNA extracted

prepared

by

in the series on from

MN-phage.

202

R. PADMANABHAN,

R. WU AND

V. C. BODE

annealing the left or right half-molecule of the nuclease-altered DNA and the oomplementary half-molecule of untreated DNA, suggested that MN damaged only the right-hand cohesive end (Gillin & Bode, unpublished observations). In the present work we utilized the techniques developed by Wu (1970) and Wu & Taylor (1971) to sequence the single-stranded ends of X DNA. Complementary bases

were incorporated by repair synthesis at the single-stranded ends of DNA. The sequencesof these bases,in DNA extracted from control and fromkEN-treatedlambda heads, were compared. Our data indicates that MN attack takes place primarily, if not exclusively, at the right-hand cohesive end, removing four nucleotides from its 5’-terminus.

2. Materials

and Methods

(a) Micrococcal nuclease treatment Lambda heads and tails were prepared as described by Bode & Gillin (1971) and Harrison (1972). The purified heads were obtained at a concentration of about 2 x 10ll/ml. in a buffer containing 0.01 M-Tris* HCl (pH &O), O-01 as-putresciue*HCl, 10 mg bovine serum albumin/ml., 0.2 mm-EDTA and about 25% sucrose. For treatment with micrococcal nuclease, 0.05 vol. of 1 M-Tris*HCl (pH 9.0) and 0.02 vol. of O-1 M-CaCI, is added. Micrococcal nuclease (Worthington 7500 p/mg) was added to half of the preparation (MNtreated) ; the other half (control) was incubated without enzyme and carried through every subsequent step in a manner identical to the treated heads. Two pools of DNA, A and B, were used in these experiments. The MN-DNA in pool A was derived from lambda heads treated with amounts of nuclease which varied between 20 to 40 units/ml. for times which varied from 30 to 60 mm at 37’C. The MN-DNA in pool B was derived from heads uniformly treated for 30 min at 37°C with 30 units/ml. of enzyme. In every case, saturating levels of micrococcal nuclesse were used (Bode & Gillin, 1971). After incubation, the reaction was terminated by chelating the Ca2+ with a twofold excess of EDTA, and excess purified phage tails were added. Head-tail joining proceeded for at least 24 hr at 5°C before the resulting phage were purified on a CsCl gradient and the DNA extracted (Gillin & Bode, 1971). Sterile glassware and buffers were used routinely throughout the preparation. (b) Nucleotide incorporation and sequence analyak The methods of repair synthesis, enzyme degradation and analysis were those described by Wu (1970) and Wu & Taylor (1971).

3. Results (a) Repair-type synthesisat cohesiveends DNA polymerase I catalyzes the incorporation of nucleotides at the single-stranded ends of X DNA (Wu & Taylor, 1971). Labeled deoxynucleotides were thus incorporated to complete

the duplex

at the ends of MN-DNA

and control

DNA.

The

5’-

terminal ends normally extend 12 basesbeyond the 3’-ends and serve astemplates for this repair-type synthesis. If a portion there will be a deficit in the number

of the 5’-terminal ends are absent in MN-DNA, of nucleotides incorporated. Conversely, if a

portion of the 3’-terminal ends are absent in MN-DNA, there will be an excessin the number

of nucleotides

incorporated.

Finally,

there may be compensating

losses from

both the 5’- and 3’-ends. There

are 10 pGi,

10 PC, 2 pA and 2 pT residues

t The nucleotides pG, PC, pA, pT and oligonucleotides bonucleotides. For simplicity, the prefix “d” is omitted,

added to the 3’-termini of normal h mentioned in this Paper are all deoxyri-

ARRANGEMENTOFDNAINLAMBDA

PHAGE

203

HEADS

TABLET Incorporation

h :DNA

of deoxynucleotides at X DNA

single-stranded

Residues incorporated per molecule of DNA

Deoxynucleoside triphosphetes

Preperation

ends

$2

pA

i;G

,T

-Control

DNA

A

dCT;,

dATP,

dGTP,

dTTP

10.0

2.0

-

-

Control

DNA

B

dCT+,

dATP,

dGTP,

dTTP

9.7

1.7

-

-

A

dCT$,

dATP,

dGTP,

dTTP

‘7.8

1.6

-

-

B

dCT$,

ddlTP,

dGTP,

dTTP

7.7

1.2

-

-

MN-DNA MN-DNA Control

DNA

A

dCTP,

dkTP,

dGT$,

dTTP

-

1.8

9.8

1.9

Control

DNA

B

dCTP,

dATP,

dGT;,

dTTP

-

1.8

9.8

1.8

MN-DNA

A

dCTP,

dkTP.

dGT:,

d+TP

-

1.4

10.3

1.6

MN-DNA

B

dCTP,

dkTP,

dGT?‘,

dTTP

-

1.2

9.9

1.3

Standard conditions for inoorporation have been described previously (Wu, 1970). Incorporation of redio8ctivity res,ohed 8 pleteeu after 6 hr inoubetion. The values in the Table are based on measurements sfter 8 hr incubetion. The results are expressed 8s the number of labeled nualeotides incorporated into one molecule of X DNA. The symbols * and . over the deoxynucleotides denote slP- and sH-labeled compounds, respectively.

(a)

Ends

of control

X DNA

after

repoir 5’

KCCGCCGCTGGAIO;’ GGGCGGCGACCT 5’ Left -hand (b) Ends

end

of A DNA

Right-hand from micrococcal

(CCCGCCGCTGGAIO~’ GGGCGGCGACCT 5’ Left -hand

CCCGCCGCTGGA ~,o(GGGcGGCGACCT)

end

nucleose

treated heads

after

end

repair 5: CCCGCCGC L$(GGGCGGCt) Right-hand

end

Fm. 1. The cohesive end sequenoes of oontrol and micrococcal nuoleese treeted A DNA. The bases in parenthesis at the 3’ tern&i 8re those incorporated during repsir synthesis. Approximately 48,000 base pairs separate the end sequences thet 8re shown. The dssh, which is usuelly used between nucleoaides to represent a phosphete group, is omitted from this Fig. for simplicity.

DNA isolated from phage particles (Wu & Taylor, 1971). Control DNA was very similar to normal mature DNA both in the amount and in the identity of nucleotides accepted (Table 1) ; MN-DNA accepted fewer total nucleotides-2.3 less pC residues, 0.5 less residues of each pA and pT and a normal number of pG residues. In view of the known sequence of h DNA cohesive ends (Fig. l), the data suggest that the terminal tetranucleotide sequence, ApGpGpT, normally present at the right-hand 5’-terminus, is absent from at least 50% of the MN-DNA and therefore could not serve as template for the incorporation of the complementary ApCpCpT.

204

R.

PADMANABHAN,

R.

WU

(b) Nearest-neighbor

AND

V.

C.

BODE

analysis

To provide further evidence that the sequence of incorporated bases, ApCpCpT, is absent when MN-DNA serves as a template, a partial nearest-neighbor analysis was carried out (Table 2). Since the total number of each 3aP-labeled nucleotide incorporated into the cohesive ends of a h DNA molecule is known, the results are expressed as TABLET Nearest-neighbor Labeled nuoleoside triphosphate

analysis of nucleotides added to the cohesive ends of h DNA Number of nearest-neighbor dinuoleotides In normal Control DNA MN-DNA cohesive ends A B A B

Isolated 3’ deoxynuoleotide

Derived sequence

PP%

TP AP cp GP

TPG APG CPG GPG

0.2 1.0 3.8 4.8

0.5 0.8 4.0 4.7

0.3 0.9 3.9 4.9

0.4 0.8 3.8 5.0

0.4 0.7 3.7 5.2

PPhC

TP AP CP GP

TPC APC CPC GPC

1.1 1.2 3.7 4.0

1.3 1.0 3.8 3.8

1-l 1.2 3.8 3.9

l-2 0.3 2.7 3.8

1.2 0.1 2.6 4.1

The results are expressed as the number of the indicated dinuoleotide sequences in the repaired ends of one h DNA molecule. The oalculetion was based on 8 knowledge of the total number of the particular 3aP-lebeled nuoleotide residues incorporated into the cohesive en& of one molecule. The values are the average of 3 sets of experiments. The number of nearest-neighbor dinucleotides in normal cohesive ends is that reported by Wu & Taylor (1971). * symbol over deoxynucleotides denotes s2P-labeled compounds.

the number of each indicated nearest-neighbor sequence, rather than as a frequency. The nearest-neighbors to 3aP-labeled pG residues are the same in MN and control DNA. Furthermore, they correspond in amount to the number expected from the known sequenoes of X cohesive ends. When the same type of analysis is performed for 32P-labeled pC residues, MN-DNA molecules differ from control DNA by the absence of O-9 ApC sequence and l-1 CpC sequence. TABLE

3

$-end group analysis of nucleotides added to h DNA

cohesive ends

Nuoleosides h DNA Control Control MN-DNA MN-DNA

DNA DNA

Preparation A B A B

T

A

C

G

0.8 0.8 0.2 0.1

0.2 0.1 0 0.3

0.9 1.1 0.9 0.9

0.1 0.2 1.0 0.8

The DNA was labeled at the cohesive ends by using all 4 3H-lebeled deoxynuoleotides during the enzymio repair synthesis (Wu, 1970). The number of nuoleoside residues per mole&e is caloulated from the total counts recovered as both nucleotides and nuoleosides after enzymic degradation of the labeled DNA and from the total number of residues of eech nuoleotido incorporat)ed during repair synthesis,

ARRANGEMENTOFDNAINLAMBDAPHAGE

HEADS

205

(c) r-end group analysis Although the pA and pT incorporation data alone did not clearly show that every molecule of MN-DNA had all four bases removed from the right-hand single-stranded terminus, the nearest-neighbor analysis discussed above indicated that approximately 90% of the MN-DNA molecules failed to serve as template for the incorporation of the ApCpCpT sequence. An analysis of the 3’-end group of the incorporated nucleotides further supports the conclusion that nearly all molecules of MN-DNA lack 5’-terminal tetranucleotide at the right-hand end (Table 3). In control DNA, the 3’-end nucleosides were the expected ones: dC and dT. In MN-DNA, they were dC and dG in essentially unit amounts, as predicted if the ApCpCpT sequence is completely missing from the right-hand end of all the molecules.

4. Discussion Since dG is the 3’-terminal residue of both strands of X DNA before DNA polymerase catalyzed repair synthesis (Fig. I), the observation of normal pG incorporation, but decreased total incorporation, of the other three nucleotides (Table 1) reinforce the previous conclusion (Gillin & Bode, 1971) that the 3’-end of MN-DNA are not the site of MN-catalyzed degradation. Therefore, we will consider the results in terms of alterations in the 5’ single-stranded ends. The incorporation data alone is consistant with several different models for the structure of MN-DNA. However, in combination with all the other data, it appears that the fractional decrease in pA and pT incorporation was low and should be taken as a unit loss of each residue. Nearest-neighbor analysis supports this assumption. The sequence ApC occurs only in the repaired right-hand cohesive end and is complementary to the third and fourth residue. The results (Table 2) indicate that this sequence, and hence the terminal tetranucleotide, is absent in essentially every molecule of MN-DNA. This conclusion is strengthened by the 3’-end group analysis (Table 3) in which dG in MN-DNA replaced dT in control DNA, and no excess over one dC or background levels of dT were found. We cannot rule out the possibility that the four bases are incompletely removed from the righthand end, or that a terminal G residue may also be removed from the left-hand end in 10% of the MN-DNA molecules. The possibility of up to 10% heterogeneity in the MN-DNA population cannot be ruled out even after complete sequence analysis of the cohesive ends of MN-DNA. However, these uncertainties do not alter our main conclusion that MN treatment of tailless heads removes four bases from the right-hand end of each A DNA molecule. No DNA degradation occurs if a tail is attached to the head before nuclease treatment (Bode & Gillin, 1971). Therefore we conclude that in the phage head X DNA is packed withits right-hand end exposed at the tail attachment site. The location of the left end cannot be inferred from our observations. It could be packed near the tail attachment site ; if so, the end nucleotides are probably not exposed. We suppose that the high MN concentration and long incubation times have minimized any effects of base sequence on the action of the enzyme. Little & Gottesman (1971) present data which suggest that X DNA molecules with only one cohesive end can be packaged in X heads but that only those heads containing a DNA molecule with a right-hand end subsequently join with a tail. MN-treated heads join quantitatively with tails in vitro (Bode & Gillin, 1971). If the right-hand end is essential for head-tail joining, then the first four bases of the 5’ terminated strand are not critically involved in the joining process itself.

206

R.

PADMANABHAN,

R.

WU

AND

V.

C.

BODE

Sharp, Donta & Freifelder (1971) examined the fragments of DNA molecules injected into bacteria by X-irradiated X phage. Presumably infection proceeds normally with these particles but stops at the first double-stranded break. They concluded that the phage population is divided equally between those particles which inject their DNA beginning with the left-hand end and those which inject beginning with the right-hand end. If their conclusions are correct, they cannot be explained on the basis of a random polarity of the packaged molecules. The results presented here strongly indicate that, in all particles, the right-hand end is at the tail attachment site. This further suggests that the arrangement of DNA is uniform in all h heads. The physical properties of MN-DNA and the biological consequences of the MNcatalyzed alteration of the cohesive ends were described m the previous papers of this series. They are summarized again in Table 4. As was indicated above, the four bases, TABLE

4

Properties after removal of four bases I MN-he&e (a) Tail II

III

joining

MN-p-0 (a) Absorption to bacteria (b) Injeotion of DNA (c) Plaque forming efficienoy (d) Frequenoy of lysogenization (e) Ratio of efficienoy of plating

Normal

at 32’C

to that

MN-DNA (a) Single-&ended moleouler weight (b) Transforming activity of DNA (whole or half molecules) (o) Rate of H-bonded circle formation at 10°C (d) T= of H-bonded oircles (e) In w&o oovalently closed cirole formation (speoies I) at 37’C

at 40°C

Normal Normal 6 to 15% of control 10% of control 6: 1 Normal Normal 7 x faster 25°C lower 10%

than than

oontrol control

of control

ApGpGpT, are not essential for head-tail joining or for DNA injection. The low frequency of plaque formation and of lysogeny after infection with complete phage particles formed by joining MN-heads with tails in vitro, is attributed to the failure of MN-DNA to cyclize efficiently, thus preventing repair in v&o. It may be pointed out that, even after the removal of the four nucleotides ApGpGpT to reduce the length of the right-hand cohesive end to an eight nucleotide-long G-C run, MN-treated DNA still can form stable hydrogen-bonded circles with the left-hand cohesive end. This is consistent with results obtained from direct binding studies. It was found (Wu, 1972) that the isolated G + C?rich octanucleotide complementary to the residual right-hand sequence, can form a stable duplex with the cohesive end of intact h DNA. The extent of binding was more extensive, however, with longer oligonucleotides such as the nona- or dodecanuoleotide. Now that its structure is reasonably well established, MN-DNA could be useful in studies of DNA repair and function. Ligase treatment after cyclization should yield circular molecules with a defined four base gap. This gap would occur in the r-strand which is the one normally transcribed in this region of the molecule.

ARRANGEMENTOFDNAINLAMBDAPHAGEHEADS This work was supported by grants and GB 26163) and the Public Health

from the National Science Foundation Service (GM 18182 and GM 18887).

207 (GB

31485X

REFERENCES Bode, V. C. & Gillin, F. D. (1971). J. Mol. Biol. 62, 493. Donelson, J. & Wu, R. (1972). J. Biol. Chem. 247, in the press. Gillin, F. D. & Bode, V. C. (1971). J. Mol. Biol. 62, 503. Harrison, D. (1972). A Study of Bacteriophage Lambda Heads and Tails, Thesis, University of Maryland, U.S.A. Little, J. W., L Gottesman, M. (1971). In The Bacteriophage Lambda, p. 371, ed. by A. D. Hershey, New York: The Cold Spring Harbor Laboratory. Sharp, J. D., Donta, S. & Freifelder, D. (1971). Virology, 43, 176. Wu, R. (1970). J. Mol. Biol. 51, 501 Wu, R. (1972). Nature, 236, 198. Wu, R. & Taylor, E. (1971). J. Mol. Biol. 57, 491.