An alkaline sucrose gradient analysis of the mechanism of nuclear DNA synthesis in the yeast Saccharomyces cerevisiae

Abstract

Using alkaline sucrose gradients the mechanism of DNA synthesis has been investigated in both log-phase and synchronised cultures of the yeast Saccharomyces cerevisiae. DNA synthesis proceeds via a heterogeneous population of single-stranded intermediates between 7 and 60 x 10(6) daltons in size. The size of these molecules and a comparison of their behaviour in log-phase and synchronised cultures suggests they are nascent or completed replicons. The progressive increase in molecular weight of these intermediates during S in synchronous cultures was used as a measure of the rate of DNA synthesis per single strand. During the first half of the period of DNA synthesis in the culture, the observed rate of elongation was 0.82 x 10(6) daltons/min. Later in S, an apparent increase in rate was detected, but this may have reflected the joining of completed replicons. In our gradients the pattern of DNA synthesis in the cell cycle mutants cdc2 and 6, thought to make incomplete or faulty DNA at the restrictive temperature (Hartwell, 1974), closely resembled that of the wild-type.