Regulation of CDC9, the Saccharomyces cerevisiae gene that encodes DNA ligase.

Abstract

We have cloned CDC9, the structural gene for Saccharomyces cerevisiae DNA ligase, and investigated its transcriptional regulation both as a function of cell cycle stage and after UV irradiation. The steady-state level of DNA ligase mRNA increases at least fourfold in late G1, after the completion of start but before S phase. This high level of CDC9 mRNA then decays with an apparent half-life of ca. 20 min and remains at a low basal level throughout the rest of the cell cycle. The accumulation of CDC9 mRNA in late Gl is dependent upon the completion of start but not the CDC7 and CDC8 functions. Exposure of cells to UV light elicits an eightfold increase in DNA ligase mRNA levels.The yeast Saccharomyces cerevisiae offers the opportunity to combine sophisticated genetic and biochemical analyses to the study of eucaryotic cell division. In attempting to elucidate the molecular events of the cell division cycle, researchers have asked whether yeast genes are expressed periodically during the cell cycle. Initial studies on the activities of various enzymes in cultures of synchronously dividing cells did show significant periodic fluctuations in activity (21). However, subsequent work which examined synthesis of over 200 major yeast proteins found no evidence for periodic synthesis; instead, the proteins analyzed showed exponential synthesis during the cell cycle (7). It was suggested that the previously observed activity modulations were the result not of changes in enzyme synthesis but rather of periodic changes in regulatory molecules affecting enzyme activity (8). The only exceptions noted were the histones; these proteins did show a cell cycle periodicity in synthesis, being synthesized maximally in early S phase (7). Expression of the yeast histone genes is now known to be cell cycle regulated by both transcriptional and posttranscriptional controls (10, 13). To our knowledge, the histone genes and the homothallism gene HO (24) are the only identified yeast genes known to be subject to mitotic cell cycle regulation at the level of synthesis of gene products. However, a recent examination of ca. 900 5. cerevisiae proteins revealed eight other proteins of unknown identity which appeared to be synthesized periodically in the cell cycle (19).Of the many yeast cdc (cell division cycle) mutants isolated, cdc9 is one of the few in which the biochemical defect has been identified. Johnston and Nasmyth demonstrated that cdc9 mutants are defective in DNA ligase activity (17). While this manuscript was in preparation, we became aware that CDC9 was recently cloned and shown to encode a DNA ligase by virtue of its ability to complement the Schizosaccharomyces pombe cdcl7 mutation, which lies in the DNA ligase structural gene of that organism (1,25). We report here the independent molecular cloning and transcriptional characterization of CDC9.* Corresponding author.DNA ligase functions in at least three processes in vivo: DNA replication, repair, and recombination. Since DNA replication is periodic and levels of DNA repair and...

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

“…This result leads us to conclude that whatever the nature of the inducing signal, it too shows a dose-dependent response to DNA damage. Some yeast genes have been shown to be regulated during the mitotic cell cycle, e.g., CDC9 (26), although the vast majority do not show such regulation (11). It is possible that RAD54 shows this kind of regulation, because it is known that cells exposed to X-rays characteristically show a division delay during G2, after DNA replication.…”

Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase.

“…romyces cerevisiae, not only are genes encoding the enzymatic machinery for DNA synthesis cell-cycle regulated [for example POLl, DNA polymerase I (Johnston et al 1987); CDC9, DNA ligase (Barker et al 1985;Peterson et al 1985)], but so are many of the enzymatic activities involved in the production of the dNTP precursors needed for DNA synthesis [for example, CDC8, thymidylate kinase (White et al 1988); CDC21, thymidylate synthase IStorms et al 1984) and ribonucleotide reductase (Lowden and Vitols 1973)]. Several other genes associated with DNA metabolism are also cell-cycle regulated; these include histones (Hereford et al 1981); HO, an endonuclease involved in mating type switching; SW15, a regulator of mating type switching (Nasmyth 1987); and RAD6 (Kupiec and Simchen 1986).…”

Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast.

“…To confirm the periodic transcription of the CDC9 message, initially reported by Peterson et al (1985), and to establish its precise timing, three different synchronisation methods were used. Firstly, S. cerevisiae cells were synchronised using the feed -starve procedure of Williamson and Scopes (1962) (Figure 1).…”

“…In view of this, we decided to investigate whether the similarities between DNA ligase in the two yeasts would extend to their regulation and, in particular, whether both would be periodically transcribed. Peterson et al (1985) used synchronous cultures prepared using a-factor and size selection in sucrose gradients to infer that CDC9 may be periodically transcribed. We have now confirmed this by using elutriation and feed -starve synchrony, and have gone on to accurately time CDC9 transcription with respect to the S phase and histone transcription.…”