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

Abstract

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...

Physical map of bacteriophage BF23 DNA: terminal redundancy and localization of single-chain interruptions

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Phased diploid genome assembly with single-molecule real-time sequencing

“…Shearing of the DNA was performed either with G-tubes (Covaris®) or by passage through a small bore needle 37 to average size of 15 kbp to 40 kbp. The needle method was used during an evaluation of shearing techniques.…”

Double-strand breaks from single photochemical events in DNA containing 5-bromouracil

“…It has been reported that double-strand breaks may be produced at sites of preexisting single-strand nicks by enzymes and shear forces (Hayward, 1974). To investigate the possibility that the observed double-strand breaks were artifacts of such action, E. coli AB2497 cells with about 6% thymine replaced by bromouracil were grown in aspartate medium to reduce the DNA content to < 1.3 genomes/cell.…”