Preferred breakage points in T5 DNA molecules subjected to shear

VIROLOGY

5%.

Preferred

11-14 (1966)

Breakage

ELIZABETH Carnegie

Institution

Points BURGI,

in T5 DNA

Molecules

A. D. HERSHEY,

of Washington,

Genetics Research Accepted August

AND

Unit,

Subiected

LAURA Cold Spring

to Shear

INGRAHAM Harbor,

New York

30, 1966

T5 DNA molecules subjected to critical rates of shear break into two pieces measuring 0.6 and 0.4 of the original molecular length. The fragments of length 0.6, isolated and subjected to a higher rate of shear, break into two pieces measuring 0.4 and 0.2 of the original molecular length. The molecules must contain two to four sites of preferred breakage at prescribed locations. Preferred breakage points have not been detected and may be absent in DNA’s of some other species. RESULTS

INTRODUCTION

DNA molecules from phages T2 and lambda, under critical rates of shear, break near their centers of length, according to theoretical expectation (Hershey and Burgi, 1960; Levinthal and Davison, 1961; Burgi and Hershey, 1961, 1963; Kaiser, 1962). DNA from phage T5 behaves differently, in a manner suggesting that the molecules contain acentric points of preferred breakage (Hershey et al., 1963b). The results presented below show that there are at least two such points per molecule and that they are similarly situated in all or most of the molecules. MATERIALS

AND

METHODS

T5 DNA, usually labeled with tritium or radiophosphorus, was prepared by the phenol met,hod from a heat-stable (st) mutant of the phage (Hershey et al., 1963b; Burgi, 1963). For the preparation of H3labeled stocks, labeled uracil was substituted for the uridine formerly used. Breakage under shear was accomplished by stirring DNA solut,ions in buffered 0.6 M NaCl for 30 minutes at 5°C (Hershey et al., 1962). Breakage products were fractionated by chromatography (Mandell and Hershey, 1960) and analyzed by sedimentation through density gradients of sucrose (Burgi and Hershey, 1963).

Primary Breakage Products When T5 DNA is stirred at speeds just sufficient to break part or all of the DNA, breakage products of two sedimentationrate classes are invariably seen (Fig. 1). The relative amounts of the two products, and their sedimentation rates, do not depend significantly on the fraction of the molecules broken, indicating that the first and the last molecules to break yield the same products, and that both products are derived from the same molecules. If the original DNA and its breakage products are linear structures whose only hydrodynamically important difference is length, the lengths can be calculated from the sedimentation rates (Burgi and Hershey, 1963). On this basis, the data of Fig. 1 assign to the breakage products the lengths 0.6 and 0.4, measured in relation to the unbroken DNA of unit length (molecular weight 75-80 million; Burgi and Hershey, 1963). Since the areas under the two sedimenting peaks also correspond very nearly to a 60:40 ratio, the interpretation of the sedimentation rates as measures of a length and mass difference is correct. Molecules of T5 DNA therefore contain sites of preferred breakage at points distant about 40 % of the molecular length from one or both ends. Not all molecules break at the prescribed 11

12

BURGI,

HERSHEY

AND

INGRAHAM

cluting at lower salt concentrations than the longer ones. Xost of the recovered fragments belong t,o one class or the ot,her, though neither class is perfectly homogeneous, and the exact’ compo&ion of each class remains unknown. Figure 2 shows the sedimentation character of a fraction consisting mainly of 0.6 unit fragmenbs.

20

15 E % IO ; : P; 5

0

20

30 Distonce

(mm)

FIG. 1. Sedimentation pattern of T5 DNA partly broken under shear. Solid line: PWabeled DNA stirred at 10 pg/ml and 1050 rpm. Broken line: H3-labeled marker DNA stirred separately. Left to right, unbroken DNA, 0.6 and 0.4 unit fragments. Centrifugation, 4.3 X lo9 rpm2 hr. Distance measured from the meniscus.

positions, because chromatographic analysis reveals small numbers of fragments of diincluding half lengths. verse lengths, Whether the exceptional fragments arise from exceptional molecules, or result from accidents of breakage, we have not yet determined. Secondary Breakage Products When a sample of partly broken T5 DNA is restirred at-a higher speed sufficient to produce additional breaks, the fragments 0.6 unit long disappear, and most of the DNA now sediments as a single band at the rate characteristic of fragments 0.4 unit long. This behavior confirms the postulated length difference of the initial breakage products, and suggests that the 0.6 unit fragments themselves break acentrically. Breakage of the 0.6 unit fragments is best seen after isolating them. On passage through a column of methylated serum albumin, a partly broken sample of T5 DNA separates into two bands corresponding to broken and unbroken fractions and, at times, the band corresponding to broken DNA is visibly double. At all times, sedimentation analysis of individual fractions shows that the 0.4 and 0.6 unit fragments are resolved, the shorter ones

% ; IO g a 5

o

e

30

20 Distance

(mm)

FIG. 2. Sedimentation pattern of isolated 0.6 unit fragments. Solid line: a single chromatographic fraction, 21% of the total DNA, isolated from the PWabeled breakage products of Fig. 1. Broken line: unfractionated H3-labeled marker DNA also shown in Fig. 1. Centrifugation 4.3 X

log rPm2 hr. 15

E 10 : f : a 2

r

I JJ

C

I

LI --J-l I

20

30 Distance

IO

(mm)

FIG. 3. Sedimentation pattern of isolated 0.6 unit fragments after further breakage under shear. Solid line: Pa*-labeled material shown in Fig. 2 restirred at 2 rg/ml. Broken line: same marker DNA as in Figs. 1 and 2. Centrifugation 5.6 X lo9 rpm2 hr. The band containing unbroken marker DNA, having sedimented nearly to the bottom of the tube, is not shown in the figure.

BREAKAGE

POINTS

When a solution containing 0.6 unit fragments, isolated as described, is stirred at a series of increasing speeds, the starting material gives way to two simultaneously generated products sedimenting at rates corresponding to 0.4 and 0.2 unit lengths, respectively. Figure 3 shows the result after complete breakage of the starting material. The masses of the two breakage products, measured as areas in the sedimentation pattern, do not exactly correspond to the lengths estimated from rates. The discrepancy can be attributed to the inhomogeneity of the starting material (Fig. 2), because the length assignment derived from sedimentation rates is supported by the chromatographic properties of the breakage products. Specifically, the 0.4 unit fragments generated on initial breakage of intact molecules, and on breakage of 0.6 unit fragments, are chromatographically indistinguishable, whereas the fragments that sediment more slowly are also eluted from the columns at lower salt concentrations. Therefore the 0.6 unit fragments contain sites of preferred breakage at points distant about 40% of the original molecular length from one or both ends. DISCUSSION

Our results show that T5 DNA molecules contain at least two preferred breakage points at the same or nearly the same location in all or most of the molecules. The acentric breakage we observe implies that the molecules contain weak spots, which could be distributed in any of at least three ways: (1) two spots at positions 0.4 and 0.6 of the total molecular length measured from either end; (2) two spots at positions 0.4 and 0.8 measured from a specified end; or (3) four spots dividing the molecule into 5 equal segments. The individual spots must be short compared with 20 % of the molecular length, and there cannot be more than four unless they are clustered, or unless others lie very close to the original molecular ends. (A weak spot lying too far from the center of a molecule or fragment subjected to shear would not in general reveal itself as a site of preferred breakage.) It is likely, though not logically necessary,

IN DNA

13

that weak spots in prescribed locations signify prescribed nucleotide sequences at those locations. If so, our results suggest that the molecules are not circularly permuted in the sense postulated for T4 DNA (Streisinger et al., 1964). In fact, Thomas and Rubenstein (1964) have shown that nucleotide sequences in T5 DNA are not circularly permuted. Our own evidence in this connection is somewhat trivial since it leaves open the possibility of a segmental permutation compatible with hypothesis (3) of the preceding paragraph. In earlier work, the DNA’s of T2 and T5 were found to exhibit equal fragility per unit length under shear (Hershey et al., 1962). This fact and results reported here might suggest that both DNA’s contain weak spots, which do not reveal themselves as preferential breakage points in T2 DNA precisely because the molecules are circularly permuted (Thomas and Rubenstein, 1964). That interpretation seems unlikely because (1) lambda DNA breaks under shear at centrally clustered points (Burgi and Hershey, 1963) ; (2) lambda DNA molecules are not circularly permuted (Kaiser, 1962; Hershey et al., 1963a); and (3) lambda DNA also matches T2 DNA in fragility per unit length (Burgi and Hershey, 1963). Possibly all three DNA’s contain weak spots, which in lambda happen to be centrally clustered. More likely only T5 DNA contains singular weak spots, which weaken the molecules so little that in fact they sometimes break centrally. A small overall effect could have been missed in the comparison with T2 DNA. Concerning the structure of the weak spots one can only list hypotheses. (1) They may be linkages of unknown type, nearly as strong as the known covalent bonds. (2) They may be points at which one of the polynucleotide chains of the helix is interrupted (Davison et al., 1964). (3) They may be regions bounded by interruptions in both strands, regions within which only the secondary forces responsible for helical structure hold the molecules together. (4) As suggested to us by C. A. Thomas, Jr., they may be rare nucleotide sequences within which secondary structure is char-

acteristically weak if, as seems likely, secondary structure cont’ributes appreciably to the overall strength of the molecule. It may be noted that T5 DKA does contain int,errupted polynucleotide chains (Davison et al., 1964; also our preparations). Whether or not the gaps are responsible for the observed breakage characteristics remains to be ascertained. REFERENCES BURGI, E. (1963). Changes in molecular weight of DNA accompanying mutations in phage. Proc. Natl. Acad. Sci. U.S. 49, 151-155. BVRGI, E., and HERSHEY, A. D. (1961). A relative molecular weight series derived from the nucleic acid of bacteriophage T2. J. Mol. Biol. 3, 45%

472. BVRGI, E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309321. D.~VISON, P. F., FREIFELDER, D., and HOLLOWAY, B. W. (1964). Interruptions in the polynucleotide strands in bacteriophage DNA. J. Mol. Biol. 8, l-10. HERSHEY, A. D., and BURGI, E. (1960). Molecular homogeneity of the deoxyribonucleic acid of phage T2. J. Mol. Biol. 2, 143-152. HERSHEY, A. D., BVRGI, E., and INGRAHAM, L.

(1962). Sedimentation coefficient and fragility under hydrodynamic shear as measures of molecular weight of the DNA of phage T5. Biophys. J. 2, 423-431. HERSHEY, A. I)., Bunor, E., and INGRAHAM, L. (1963a). Cohesion of DNA molecules isolat,ed from phage lambda. Proc. Natl. Acad. Sci. U.S.

49, 748-755. HERSHEY, A. I)., GOLDBERG, E., BURGI, E., and INGRAHAM, L. (1963b). Local denaturation of DNA by shearing forces and by heat. J. Mol.

Biol. 6, 230-243. KAISER, A. D. (1962). The production of phage chromosome fragments and their capacity for genetic transfer. J. Mol. Biol. 4, 275-287. LEVINTHAL, C., and DAVISON, P. F. (1961). Degradation of deoxyribonucleic acid under hydrodynamic shearing forces. J. Mol. Biol. 3, 674-

683. MANDELL, J. D., and HERSHEY, A. D. (1960). A fractionating column for analysis of nucleic acids. Anal. Biochem. 1, 66-77. STREISINGER, G., EDGAR, R. S., and DENHARDT, G. H. (1964). Chromosome structure in phage T4. I. Circularity of the linkage map. Proc. Natl. Acad. Sci. U.S. 51, 775-779. THoMas, C. A., JR., and RUBENSTEIN, I. (1964). The arrangements of nucleotide sequences in T2 and T5 bacteriophage DNA molecules. Biophys. J. 4, 93-106.