The activity of mammalian polynucleotide ligase on x-irradiated DNAs

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.