Local denaturation of DNA by shearing forces and by heat

Hershey, Alfred Day; Goldberg, Edward B.; Burgi, Elizabeth; Ingraham, Laura J.

doi:10.1016/s0022-2836(63)80072-8

Cited by 60 publications

(22 citation statements)

References 11 publications

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“…Several recent reports have supported the possibility that at least some of this variability resulted from contamination with single-stranded or denatured DNA (ssDNA) (3,4). This seems likely to have arisen during isolation and storage of the DNA and is probably related in part to the rigidity of the double helical molecule, long recognized as being vulnerable to breakage and partial denaturation as the result of exposure to shearing forces (5,6).…”

Section: Introductionmentioning

confidence: 99%

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Steinman¹,

Deesomchok²,

Spiera³

1976

J. Clin. Invest.

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Section: Introductionmentioning

confidence: 99%

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Steinman¹,

Deesomchok²,

Spiera³

1976

J. Clin. Invest.

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“…This property has been utilized for crude fractionation of some viral genomes (such as X DNA) in which segmented base composition permits the right and left halves and even smaller segments to be physically separated (3). Under certain conditions the breakage events can be associated with partial denaturation and the presence of singlestranded tails on the duplex products, although these can be avoided (4,5).…”

mentioning

confidence: 99%

Unique Double-Stranded Fragments of Bacteriophage T5 DNA Resulting from Preferential Shear-Induced Breakage at Nicks

Hayward

1974

Proc. Natl. Acad. Sci. U.S.A.

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Nicks within one strand of the bacteriophage T5 DNA molecule act as "weak points" for a novel kind of mechanical breakage that can be utilized for "dissecting" the genome. The products from sheared T5+ DNA include five unique double-stranded segments of the molecule and various combinations of adjacent segments. These specific fragments are not obtained after repair of the nicks with DNA ligase (EC 6.5.1.1). The duplex fragments and most of their single-stranded components have been separated, identified, and mapped by means of agarose gel electrophoresis. Even the complementary strands of the unique fragments separate in agarose gels; hence, there are now three useful classes of DNA fragments available from T5: the natural r-strand fragments, their complements from the normally intact I strand, and the corresponding duplex segments. By summing the apparent molecular weights of their single-stranded components, the unique duplex fragments from T5+ DNA can be as- All large double-stranded DNA molecules are susceptible to breakage by shear. In the case of linear viral DNA molecules the breakage products generally have a Gaussian size distribution centering at halves, quarters, etc. of the original molecular length depending on the strength of the shearing forces used (1, 2). This property has been utilized for crude fractionation of some viral genomes (such as X DNA) in which segmented base composition permits the right and left halves and even smaller segments to be physically separated (3). Under certain conditions the breakage events can be associated with partial denaturation and the presence of singlestranded tails on the duplex products, although these can be avoided (4, 5).As first observed by Burgi, Hershey, and Ingraham (6), the bacteriophage T5 DNA molecule breaks in a novel acentric fashion, producing fragments that sediment as several distinct peaks in sucrose gradients. The initial breakage products from T5st(O) DNA have molecular weights of approximately 60% and 40%0 that of the parent genome, and under stronger shearing conditions the larger pieces again break acentrically (6).Rubenstein (7) has shown that the DNAs from T5+ and several of its deletion mutants give different and characteristic breakage products, but he was unable to distinguish between the possibilities that the initial breakage sites exist on one particular side of the -molecule only, or are spaced equidistant from either end. Similarly, these early experiments did not establish whether the preferred breakage occurred at precisely defined positions rather than over relatively broad regions of the molecule, although Rubenstein suggested that the "weak points" may correspond to the single-strand interruptions known to exist in T5 DNA (8). These interruptions have subsequently been shown to be specific nicks that occur in only one strand of the duplex and can be repaired with DNA ligase (9, 10).In this paper I present evidence demonstrating that all of the nicks in the T5 DNA molecule can provide precise points for preferential...

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“…These studies led to the proposal of 12 alternative models. Common features of all these models were that both DNA strands were interrupted and that the total number of four interruptions existed per DNA molecule.…”

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confidence: 99%

LOCATION OF SINGLE-STRAND INTERRUPTIONS IN THE DNA OF BACTERIOPHAGE T5 ⁺

Bujard

1969

Proc. Natl. Acad. Sci. U.S.A.

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Abstract.-The positions of three single-strand interruptions in the DNA of phage T5+ have been located by electron microscopy. All three interruptions were found in the same strand. Uneven base composition along the molecule is indicated by the preferential melting of certain regions. The data suggest a model according to which (1) the first-step-transfer DNA section is separated by a single-strand interruption from the rest of the phage genome, (2) the phage carries only one such section and therefore transfers the asymmetrical DNA molecule always in the same direction into the host cell, and (3) single-strand interruptions are points of preferred breakage.Bacteriophage T5 has unusual properties not found in most other well-studied coliphages: (1) During infection, the phage transfers its DNA in a two-step process.", 2 Initially, a well-defined portion, the so-called first-step-transfer DNA,3 representing about 8 per cent of the DNA molecule, is transferred to the host cell.4 5 Protein synthesis directed by this portion of the DNA is required for subsequent transfer of the rest of the DNA molecule.6 (2) The nucleotide sequence of the linear, doubled-stranded DNA is unique, not circularly permuted.7 (3) When exposed to shear in solution, the point of preferred breakage of isolated T5 DNA is acentric.8 9 (4) T5 DNA contains single-strand interruptions in defined positions.'0 It seems pertinent to ask whether properties (1) to (3) above are directly correlated with the occurrence of single-strand interruptions in the DNA molecule.Furthermore, it would be interesting to know whether the DNA possesses a first-step transfer section at each end, and, if so, whether transfer to the host cell can be initiated from either end of the molecule. The knowledge of the exact number and positions of the single-strand interruptions within the DNA molecule might well clarify at least some of these problems.Based on shearing and heat-denaturation experiments, three alternative models for the distribution of the points of preferred breakage were proposed:8 (1) one point at 40 and one at 60 per cent of the total molecular length from either end, (2) one point at 40 and another at 80 per cent of the total molecular length when measured from one end, or (3) four points dividing the molecule in five equal segments.Abelson and Thomas'0 studied the sedimentation of native and denatured DNA in experiments made to determine the number and distribution of singlestrand interruptions in the T5 DNA. These studies led to the proposal of 12 alternative models. Common features of all these models were that both DNA strands were interrupted and that the total number of four interruptions existed per DNA molecule. Some of these models can be correlated with those proposed 1167

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Local denaturation of DNA by shearing forces and by heat

Cited by 60 publications

References 11 publications

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Unique Double-Stranded Fragments of Bacteriophage T5 DNA Resulting from Preferential Shear-Induced Breakage at Nicks

LOCATION OF SINGLE-STRAND INTERRUPTIONS IN THE DNA OF BACTERIOPHAGE T5 ⁺

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Local denaturation of DNA by shearing forces and by heat

Cited by 60 publications

References 11 publications

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Detection of anti-DNA antibody using synthetic antigens. Characterization and clinical significance of binding of poly (deoxyadenylate-deoxythymidylate) by serum.

Unique Double-Stranded Fragments of Bacteriophage T5 DNA Resulting from Preferential Shear-Induced Breakage at Nicks

LOCATION OF SINGLE-STRAND INTERRUPTIONS IN THE DNA OF BACTERIOPHAGE T5 +

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LOCATION OF SINGLE-STRAND INTERRUPTIONS IN THE DNA OF BACTERIOPHAGE T5 ⁺