Pulsed Field Gel Electrophoresis Labeling Method to Study the Pattern of Saccharomyces cerevisiae Chromosomal DNA Synthesis During the G1/S Phase of the Cell Cycle

Abstract

Yeast Saccharomyces cerevisiae is an excellent model to study eukaryotic DNA replication. Since yeast lacks thymidine kinase, it is difficult to assay DNA synthesis. In this report, a novel approach called the pulsed field gel electrophoresis (PFGE) labeling method is used to investigate yeast chromosomal DNA synthesis. In this method, yeast cells are first labeled by 32P in vivo and chromosomal DNA molecules are then resolved by pulsed field gel electrophoresis. A linear 32P labeling of chromosomal-size DNA molecules can be observed up to 100 min in an asynchronized culture. In an α-factor arresting-and-releasing synchronized culture, we observed that 32P can be rapidly taken up in the S phase. Our results show that all of the chromosomes are labeled at approximately the same time (within a 15-min interval), suggesting that the temporal order of all chromosomal DNA synthesis is synchronized in the S phase. Cell cycle blockers were used to arrest the yeast cultures to study DNA synthesis that coincides with cell cycle analysis. Our results show that α-factor and hydroxyurea block the cell cycle at the late G1 and S phases, respectively, resulting in the failure of DNA synthesis. Nocodazole blocks the cell cycle after the S phase and thus shows no effect on chromosomal DNA synthesis. Since the PFGE labeling method is highly specific to chromosomal DNA synthesis, we used it to examine the DNA synthesis pattern in various cell cycle mutants. All of the G1/S cdc mutants tested, cdc4, 6, 7, 8, 17, 2, and 9, cannot label the chromosomal size of DNA molecules; but the G2/M cdc mutant cdc13 can. Specific findings are (i) cdc9 and cdc7 mutant cells do not synthesize chromosomal DNA molecules, although some 32P incorporation could be observed at nonpermissive temperature; (ii) cdc6 is confirmed to be defective in DNA synthesis; and (iii) all three yeast polymerases are defective in DNA synthesis, suggesting their role(s) are in the elongation step. Thus, the use of the PFGE labeling method combined with genetic studies in yeast would facilitate exploration of the mechanism(s) of DNA replication in eukaryotes.