The minichromosome maintenance (MCM) proteins, together with the origin recognition complex (ORC) proteins and Cdc6, play an essential role in eukaryotic DNA replication through the formation of a pre-replication complex at origins of replication. We used a yeast twohybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by a highly conserved C 2 HC zinc finger and a putative histone acetyltransferase domain. Biochemical studies confirmed the interaction between MCM2 and HBO1 in vitro and in vivo. An N-terminal domain of MCM2 is necessary for binding to HBO1, and a C 2 HC zinc finger of HBO1 is essential for binding to MCM2. A reverse yeast two-hybrid selection was performed to isolate an allele of MCM2 that is defective for interaction with HBO1; this allele was then used to isolate a suppressor mutant of HBO1 that restores the interaction with the mutant MCM2. This suppressor mutation was located in the HBO1 zinc finger. Taken together, these findings strongly suggest that the interaction between MCM2 and HBO1 is direct and mediated by the C 2 HC zinc finger of HBO1. The biochemical and genetic interactions of MYST family protein HBO1 with two components of the replication apparatus, MCM2 and ORC1, suggest that HBO1-associated HAT activity may play a direct role in the process of DNA replication.Eukaryotic DNA replication is a tightly regulated process that is strictly coupled to cell cycle progression, ensuring that DNA is replicated only during S phase and that each origin is used only once per cell cycle. This precise cell cycle coordination is the result of both positive and negative regulation of replication origin function. Genetic and biochemical studies in yeast and metazoans suggest that the initiation of DNA synthesis is a complex, multistep process that requires the participation of many proteins (1-3). This process involves the binding of the origin recognition complex (ORC) 1 to replication origins (4, 5), the recruitment of Cdc6 and the six MCM proteins (MCM2-7; MCM for minichromosome maintenance) to form the pre-replicative complex (pre-RC) (6, 7), and the activation of the pre-RC by protein kinases to initiate DNA synthesis (8). MCM proteins were revealed to be involved in DNA replication as the result of genetic screens for mutants defective in progression through the cell division cycle (9 -12) or the replication of minichromosomes (13-15). Initial characterization of three genes, mcm2 (14), mcm3 (16), and mcm5/cdc46 (9, 17), implicated each in DNA replication and showed they were related in sequence. This family rapidly grew to encompass the Schizosaccharomyces pombe mcm4/cdc21 ϩ (18, 19) and mcm6/mis5 ϩ genes (15), and the Saccharomyces cerevisiae mcm7/cdc47 gene (20). Analysis of the complete S. cerevisiae genome sequence indicates that there are six MCM-encoding genes, and homologs of the MCM2-7 proteins have since been identified in all eukaryotes from y...

Previous experiments suggest that a key event in the commitment of cultured mammalian cells to entering S phase is a rise in activity of the transcription factor E2F. In this report, we study the role of Drosophila E2F in imaginal disc cells in vivo, by examining the distribution of the endogenous protein and studying the consequences of ectopic E2F expression. First, we find that endogenous E2F falls from high to very low levels as cells initiate DNA synthesis during a developmentally regulated Gj-S-transition in the eye disc. Second, we find that ectopic E2F expression drives many otherwise quiescent cells to enter S phase. Subsequently, cells throughout the discs express reaper (a regulator of apoptosis) and then die. Third, we find that ectopic E2F expression during S phase in normally cycling cells blocks their re-entry into S phase in the following cell cycle. Although we do not know the fate of these cells, we suspect that ultimately they are killed by ectopic E2F. Taken together, our results show that an elevation in the level of E2F is sufficient to induce imaginal disc cells to enter S phase. Furthermore, they suggest that the downregulation of E2F upon entry into S phase may be essential to prevent the induction of apoptosis.

Throughout the cell cycle of Saccharomyces cerevisiae, the level of origin recognition complex (ORC) is constant and ORCs are bound constitutively to replication origins. Replication is regulated by the recruitment of additional factors such as CDC6. ORC components are widely conserved, and it generally has been assumed that they are also stable factors bound to origins throughout the cell cycle. In this report, we show that the level of the ORC1 subunit changes dramatically throughout Drosophila development. The accumulation of ORC1 is regulated by E2F-dependent transcription. In embryos, ORC1 accumulates preferentially in proliferating cells. In the eye imaginal disc, ORC1 accumulation is cell cycle regulated, with high levels in late G1 and S phase. In the ovary, the sub-nuclear distribution of ORC1 shifts during a developmentally regulated switch from endoreplication of the entire genome to amplification of the chorion gene clusters. Furthermore, we find that overexpression of ORC1 alters the pattern of DNA synthesis in the eye disc and the ovary. Thus, replication origin activity appears to be governed in part by the level of ORC1 in Drosophila.