On the experimental distinction between ssbs and dsbs in circular DNA

Abstract

Purpose: To determine directly the minimal distance between two ssbs on complementary strands in circular DNA that are not observed as a dsb by electrophoresis. Materials and methods: 3.2kbp DNAs with cohesive overhangs of various lengths were systematically generated by a newly devised method and electrophoresed in agarose slabs. Results: At 4 C, 3.2 kbp DNA with cohesive overhangs larger than 6 bp migrated as circular DNA. The minimal overhang size for the DNA to migrate as circular DNA was larger at 25 C. Whether the DNA migrated as a circular or a linear molecule depended also on the nucleotide sequence of its overhangs, most notably at the minimal size. Conclusions: The minimal distance obtained in the present study agrees with the smaller values of previous indirect estimates. The dependence of the distance on experimental conditions suggests feasibility of obtaining the stagger-size distribution of radiationinduced dsbs.