Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency

Abstract

A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.