Nucleic Acids Research

Using mitotic cultures synchronised by a feed-starve protocol or by elutriation, we have shown that the yeast DNA polymerase I gene is periodically expressed with its transcript increasing at least 100-fold in late Gl with a peak around the Gl/S phase boundary. This is precisely the same interval of the cell cycle in which three other yeast DNA synthesis genes, CDC8, CDC9 and CDC21, have been found to be periodically expressed (White et al 1987. Expl. Cell. Res., in press). The polymerase I transscript is also regulated in meiosis, showing an overall fluctuation in level of some 20-fold, with a peak at about mid-S phase. In addition, following irradiation with 50J/m2 ultraviolet light, there was a 20-fold increase in the transcript, starting after 30 minutes and reaching a peak two hours later. These results indicate that DNA polymerase I is subject to a complex control and imply that it has a role in both DNA synthesis and DNA repair. INTRODUCTION Many of the processes of DNA metabolism are discontinuous in nature, for instance DNA synthesis occupies only a small proportion of either the mitotic cell cycle or meiosis, and DNA repair is only required occasionally. Therefore the enzymes involved in these processes could be subject to some form of regulation which allows their synthesis only when the gene product is required. In certain cases this regulation could be highly complex since a number of enzymes function in several different aspects of DNA metabolism. A prominent example is DNA ligase which acts in DNA synthesis to join Okazaki fragments and also in repair and recombination to complete the final sealing step in nicked duplex DNA. We have studied the regulation of the DNA ligase gene, CDC9, in yeast and found that in both meiosis and the mitotic cell cycle there is a brief but marked increase in CDC9 transcript levels just prior to DNA synthesis (1,2), suggesting that transcription of this gene is confined to a short interval immediately before S phase. Similarly, there is an increase in levels of both DNA ligase message and © I R L Press Limited, Oxford, England. 5017 Nucleic Acids Research enzyme after exposure of yeast cells to ultraviolet (UV) light, y-irradiation or methyl methane sulphonate (3). Another enzyme involved in many aspects of DNA metabolism is DNA polymerase. In yeast there are two species of nuclear DNA polymerase and although their respective roles are not clearly established, their biochemical properties suggest that DNA polymerase I is responsible for DNA replication(4,5,6). There have, in fact, been two previous attempts to study expression of yeast DNA polymerase, each involving the mitotic cell cycle alone (7,8). Synchronised cells were used in these experiments and total DNA polymerase activity was assayed throughout the cell cycle. No attempt was made to differentiate between species of DNA polymerase and, in summary, the results suggest that overall polymerase activity fluctuates with a peak occurring at approximately early S phase. Possibly a more direct means of examining gene expression is to follow fluctuations in level of the transcript by hybridisation with a DNA probe. This is particularly useful when several species of an enzyme exist in a cell since hybridisation is highly specific for a particular transcript. The yeast DNA polymerase I gene has recently been cloned (9,10) thus providing a specific DNA probe for monitoring its transcript. In the experiments described here, we have exploited this to examine regulation of the polymerase I gene not only in the mitotic cell cycle but also in meiosis and after UV-irradiation. MATERIALS AND METHODS Strains and Media The genotypes of the Saccharomyces cerevisiae strains were as follows; MC16 MATa leu2-3 his4-712 ade2-1 lys2-1 SUF2, NCYC239 and SK-1 are prototrophic diploids, while details of the cdc4-3 diploid MH14 have been given previously (11). Construction of the SK-1 related a/a and a/a diploids has also been described previously (1). YPD (1% Difco yeast extract, 2% Bacto peptone and 2% glucose) or YPA (1% potassium acetate in place of the 2% glucose) were used as rich media and Difco Yeast Nitrogen Base (0.67%; 0.5% glucose) as minimal medium with the appropriate nutritional supplements. Synchronisation procedures Mitotic cultures of NCYC239 were synchronised by the feed-starve protocol of Williamson and Scopes (12)and the use of the Beckman JElOX Elutriator Rotor to synchronise cells of budding yeast has been described previously (2).