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Length distributions of single-stranded DNA in Chinese hamster ovary cells

1975; Elsevier BV; Volume: 93; Issue: 2 Linguagem: Inglês

10.1016/0022-2836(75)90127-8

1089-8638

John Hozier, J. Herbert Taylor,

DNA Repair Mechanisms

The distribution of lengths of single-strand DNA in Chinese hamster ovary cells in the G1 phase of the cell cycle has been observed for various conditions of cell lysis and incubation of the lysates. The method of analysis was band sedimentation through a self-generating density gradient, a technique developed originally for the analytical ultracentrifuge, but modified here for the preparative ultracentrifuge so that measurements of sedimentation coefficients could be made under conditions that minimize shearing of the single-stranded DNA. The effect of rotor speed dependence of the sedimentation coefficient is considered in developing the relation between the sedimentation coefficient and molecular weight for this technique. Special precautions were taken to ensure that complete separation of long single strands took place upon alkaline denaturation, to preclude the possibility of anomalous sedimentation due to interstrand entanglement. Bromodeoxyuridine was incorporated into the DNA in the last round of replication. Advantage was taken of the increased sensitivity to ultraviolet irradiation for the production of single-strand breaks in DNA strands substituted with bromodeoxyuridine. After irradiation the bromodeoxyuridine-substituted strand could be completely separated from the complementary strand in alkaline sedimentation profiles without any apparent breakage in the unsubstituted strand. The conditions of lysis, chosen to minimize the degradation of DNA in the lysates, included lysis at pH 9.3 with Pronase and lysis at high pH (10.8 and 12.0). Sedimentation analysis was performed at various time intervals after incubation at 4 °C or 37 °C. Lysis and incubation at pH 12.0 produced a continuous single-strand breakdown of the DNA in the lysate. Analysis of the sedimentation profiles indicates that these alkaline-induced breaks are randomly distributed. However, lysis and incubation at pH 10.8 and at pH 9.3 with Pronase produced stable sedimentation profiles with number-average molecular weights of 1.7 × 108 and 6.0 × 107, respectively. Analysis of the single-strand DNA sedimentation profiles for these lysates indicates that the distribution of lengths of single-stranded DNA is non-random, i.e. that the distributions may represent regular subunits of chromosomal DNA structure. Suggestive evidence is presented that the approximately 60-μm units are structurally alternated in the two chains. The possible origin of the discontinuities between the subunits is also discussed.