to yeast, once the restriction point has been traversed, serum or growth factor removal does not prevent DNA synthesis. After committing to another cell cycle, yeast and vertebrate cells do not immediately initiate S phase. Instead, there is an interval, termed late GI, when cells presumably prepare for efficient and accurate genome replication. One aspect of this preparation is heightened transcription of the genes encoding enzymes required for DNA synthesis. This essay will focus on recent progress that has been made in understanding the induction of DNA synthesis gene expression in late GI in yeast. We will then consider the mechanism linking DNA synthesis gene induction to START. Finally, we will consider the evolutionary conservation of the mechanism, including its potential role in controlling DNA synthesis gcne expression in vertebrate cells.

Summary After yeast cells commit to the cell cycle in a process called START, genes required for DNA synthesis are expressed in late GI. Periodicity is mediated by a hexameric sequence, known as a MCB element, present in all DNA synthesis gene promoters. A complex that specifically binds MCBs has been identified. One polypeptide in the MCB complex is Swi6, a transcription factor that together with Swi4 also binds G1 cyclin promoters and participates in a positive feedback loop at START. The finding that Swi6 is directly involved in both START and DNA synthesis gene control suggest a model in which Swifi, activated through its participation in START, serves as the central transcription factor in coordinating late G1 gene expression. The mechanism may be conserved in all eukaryotic cells. Introduction The eukaryotic cell cycle is divided into four temporally distinct phases. After undergoing genome replication during S phase, cells continue to accumulate mass during G2 phasc, undergo mitosis and cell division during M phase, and then enter G1 phase. Then, if intracellular and extracellular conditions are appropriate for continued cell proliferation, and after accumulating sufficient mass, an irreversible decision is made in G1 to enter the next cell cycle. Alternatively, if conditions are inappropriate, GI cells withdraw from the cell cycle. In both multicellular and unicellular eukaryotes, an ability to appropriately exit and enter the cell cycle is vital to organism survival. In yeast, the G1 decision to initiate a new cell cycle is called START. Extracellular conditions that prevent START and cause withdrawal from the cell cycle include insufficient nutrient levels or the presence of mating pheromone. Once START has been traversed, nutrient deprivation or presence of mating pheromone cannot prevent another round of DNA synthesis and division. In vertebrate cells a similar decision to commit to a new cell cycle occurs in G1 and has been called the restriction pointi'). In vertebrate cells in culture, extracellular conditions that prevent restriction point traversal include serum deprivation or removal of purified growth factors. Similarly

DNA Synthesis Control from a Genetic Perspective In the budding yeast, Succharoinyces cerevisiae, classical and reverse genetics have identified many genes involved directly in DNA synthesis, such as CDC9 (DNA ligase) and CDC21 (thymidylate synthase) [see Table 1 for complete list]. In addition. a number of genes have been identified which act in late G 1 and may regulate the initiation of DNA synthesis (Fig. 1). These potential regulatory genes have been recently reviewed2).Briefly, CDC7 encodes a protein kinase that acts very close to initiation of S phase. Dbf4 interacts with Cdc7, perhaps modulating the activity or specificity of the k i n a ~ e ( ~ No ) . substrate for the Cdc7 kinase has been identified. The amino acid sequence of Cdc7 shows a potential target site for p34CdL2, a protein kinase that plays a central role during START (see below). CDC4 and CDC34 act soon after START. At nonpermissive temperatures, cdc4 and cdc34 mutants not only fail to initiate DNA synthesis, they also fail to initiate cytoskeletal aspects of the cell division cycle. MCMI, 2 and 3 probably encode proteins involved in initiation at replication origins. CDC45, 46, 47 and 54 act shortly before S phase and probably encode proteins that interact in the same process. Recent evidence suggests CDC6 also affects replication initiatiod4). Fig.1 gives the relative positions in which the genes act in late G1 (determined by genetic and physiological analysis) and creates the impression that the connection between START and the initiation of DNA synthesis is mediated by a sequence of molecular events, with each event dependent on its immediate antecedent. Results reviewed below raise the possibility that components of the START mechanism itself serve a direct role in DNA synthesis gene induction and hence S phase control.

The Mlul Cell Cycle Box (MCB), a Hexameric cisElement Controlling DNA Synthesis Gene Expression In budding yeast many, if not all, of the genes encoding proteins required for DNA synthesis are periodically expressed in late G1 (Table 1). In addition to genes directly involved in DNA precursor biosynthesis and polymerization, Table 1

START

cdc4 cdc.34

cdc7

dbf4

t

cdc45,46,4 7,54 mcm I ,2,3

Fig. 1. Genes thought to have a regulatory role in DNA replication.

includes genes thought lo be involved in replication initiation (CDC46 and DBF4) and postreplication DNA repair (RADSl and P MSI). In all cases where several DNA synthesis gene mRNAs have been measured in parallel, message levels always peaked simultaneously in late GI, suggesting that the genes arc co-regulated(2)'.The promoters of at least three of the DNA synthesis genes, CDC2 I , CDC9 and POL], confer late G 1 expression on heterologous reporter gene^(^-^), implying that most, if not all, of the increase in message levels in latc G1 is due to increased transcription. The tight co-rcgulation of the DNA synthesis genes@) suggested that they were controlled by a common mechanism and might therefore share a cominon &-element. A search of the 5' flanking sequences of CDCY. CDC21 and POLl revealed one or more copies of the hexameric sequence ACGCGT within a few hundred base pairs of the translational start codon. ACGCGT corresponds to the recognition site of the restriction endonuclease Mlul ; hence the consensus is often referred to as a Hlul Cell-cycle Box or MCB". CDC21, CUC9 and POLl promoter deletion mutations suggested that the Mlul sites were rcquired for G l/S regulation(5,6-s). An alignment of 32 Mlui-like sequences from 18 periodically expressed late G 1 genes gave a slightly more 3'-extended region of conservation, ACGCGTNPu (where N is any nucleotide and Pu is a purinc, usually A)(2). Note that the functional importance of particular MCB elenients in the context of their native promoter has only been established for CUC21(6'.Thus, the consensus may he contaminated by biologically irrelevant MCB sequences that camouflage the functionally important residues in a bonafide MCB. For example. the CDC21 promoter contains two potential MCBs. Whereas a mutation at the distal sitc greatly weakens CUC21 transcription, an identical mutation at the "'Abbreviations. ankyrin repeats (a.k.5. CdclO-Swi6 multf

DNA synthesis control in yeast: an evolutionarily conserved mechanism for regulating DNA synthesis genes?

After yeast cells commit to the cell cycle in a process called START, genes required for DNA synthesis are expressed in late G1. Periodicity is mediat...
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