Managing editors: Toshiki Tsurimoto ¹ and Hisao Masai ²

1 Faculty of Bioscience, Nara Institute of Science and Technology, Takayama, Ikoma 630-0101, Japan, ² Department of Molecular and Developmental Biology, Institute of Medical, Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan and CREST, Japan Science Technology

Precise duplication of genetic materials is central to proliferation of cells, generation and maintenance of tissues and organs as well as their inheritance to offspring. Regulation of DNA replication is under intricate intracellular programs and its initiation and elongation is strictly monitored so they are properly integrated with other events in cell cycle. Elucidation of detailed molecular mechanisms of how this process is regulated in various cells and tissues would not only provide a clue to molecular basis for malignant growth but also may lead to novel strategies by which cell growth and differentiation can be manipulated for cell and gene therapies.

Detailed enzymatic studies of DNA replication was pioneered in bacteria. These studies have established that replication of a bacterial replicon is initiated by binding of a replicon-specific initiator at the origin of DNA replication. This binding induces conformational change at the duplex DNA, which permits the loading of DNA helicase at the origin. The loading of DNA helicase involves protein-protein interactions between the initiator and helicase, and generates active replication forks to processively synthesize leading and lagging strands. Lack of origin-dependent in vitro replication assay systems in eukaryotes has hampered their rapid progress in similar studies on replication of eukaryotic chromosomes. Although analyses of yeast mutants specifically defective in G1-S transition and S phase progression suggested involvement of multiple factors for DNA replication in eukaryotes, their functions have largely remained unknown, except for some well conserved proteins for the DNA elongation machinery including DNA polymerases and single-stranded DNA binding proteins. A major breakthrough was discovery of the origin recognition complex (ORC) in budding yeast, which specifically recognizes and binds to the replication origin sequences. Subsequent studies in yeasts as well as in Xenopus egg extracts have led to identification of proteins constituting the eukaryotic replication complexes, and revealed ordered and regulated assembly of replication complexes at the replication origins. Furthermore, it has become realized that most of the yeast proteins involved in G1/S transition and DNA replication are highly conserved among eukaryotes including human, suggesting the presence of common basic mechanisms of DNA replication and its regulation. Thus, we start to understand molecules involved in mammalian DNA replication and how their functions are regulated in response to various environmental signals. Growth of mammalian cells is under strict regulation in response to various environmental factors such as growth factors, differentiation factors, contact with other cells, and DNA damaging agents, and other various stresses. G1 regulation of mammalian cells has been studied intensively, and the cyclin D-dependent kinase-Rb (retinoblastoma protein)-E2F pathway has been well established. Downstream of this pathway, another CDK, cyclin E-dependent kinase 2, may play an essential role for G1-S transition. A major question now is how this G1 regulation leads to activation of replication machinery at the replication origins. Given the rapid progress being made in this field, it will not be long before we understand the entire picture of how growth signals are integrated into intracellular signaling pathways involving G1regulators and how they ultimately lead to activation of replication origins during S phase. We thought it would be timely to summarize our current understanding of mammalian DNA replication machinery and its regulation at the G1/S transition and in the S phase. We have asked leading scientists in this field to summarize the most recent progress. Kato will describe how G1 regulators contribute to induction of S phase and also a new regulator for Cdk2-CyclinE, Jab1, which potentially plays a role in G1-S regulation. As described above, E2F is the critical transcription factor of mammalian cells which regulates expression of numerous proteins involved in G1/S transition. Ohtani will describe how E2F activity is regulated in G1 and then targets of E2F transcription factor, and finally discuss how E2F and CyclinE-dependent kinase cooperates for initiation of S phase. Quintana and Dutta will describe structures and functions of metazoan ORC, and how it would function in initiation of metazoan DNA replication. Fujita will describe another essential components for eukaryotic DNA replication complexes, Cdc6 and MCM (minichromosome maintenance), and their regulation through phosphorylation. Although replication and transcription have generally been studied by different branches of scientists, exisitence of significant interplay between them has been suggested. It has been known that transcriptionally active regions are replicated early in S phase. Some of the viral replication origins as well as cellular origin sequences are associated with transcription regulatory elements to which various transcription factors bind. Thus, coregulation of replication and transcription through transcription factors may be an important strategy to coordinately regulate cell growth and cell cycle. Murakami and Ito will discuss the possible molecular mechanisms of how transcription factors regulate DNA replication. G1 regulation ultimately leads to S phase initiation by firing replication origins. In addition to CDK, Cdc7-Dbf4 kinase complex, which was originally found in budding yeast and is now known to be conserved widely in eukaryotes, plays crucial roles in initiation of S phase. Abundance of ASK, the mammalian homologue of Dbf4, is regulated by G1-S signals and activates Cdc7 kinase only during S phase. Therefore, it may be one of the critical targets of G1-S regulation for initiation of S phase. Masai et al. will describe the structures, functions, and regulation of this novel family of kinase complexes. Once S phase is initiated, it needs to be properly completed and ensure coordinated entry into mitosis. However, cells will suffer from various insults which temporarily stall the progression of replication forks. Under these circumstances, eukaryotic cells generate intracellular signals which suspend further progression of cell cycle until any obstacle arresting the fork will be eliminated. This so called "checkpoint regulation" mechanisms appear to be amazingly conserved from yeasts to mammals. Boddy and Russell will describe the most recent progress on DNA replication and DNA damage checkpoint regulation in fission yeast as a model eukaryote. During S phase, both lagging and leading strands are synthesized in a coordinate manner with three DNA polymerases, namely alpha, delta and epsilon. At the center of this DNA chain elongation is PCNA (proliferating cell nuclear antigen) which acts as a sliding clamp for processive DNA synthesis. As Tsurimoto describes in his article, recent data indicate amazingly versatile functions of PCNA in metabolism of nucleic acids by interacting not only with DNA polymerases but also with repair factors, cell cycle machinery, and chromatin components. It probably acts as a central command office which receives various environmental signals during S phase and integrates them into their downstream signaling which appropriately modulates the structures of replication forks. Finally, it is crucial to clarify the structures of replication origins to precisely understand the events which takes place there. Mainly due to difficulties in convenient ARS assays in mammalian cells, dissection of replication origins of higher eukaryotes lagged behind that of simple monocellular eukaryotes. Todorovic et al. will summarize on structures of known mammalian replication origins and discuss requirement of cis-acting sequences for origin functions and possible protein-protein and protein-DNA interactions on them. Although most of the contents in this special issue are limited to the results of mammalian systems, directions and approaches of these studies have been profoundly affected and guided by works with yeasts and Xenopus egg extracts. Readers are referred to recent, more comprehensive review articles cited in each article in this issue for more general information. The works described by Kato, Ohtani, Fujita, Murakami, Masai, and Tsurimoto have been partly supported by a Grant-in-Aid for Scientific Research on Priority Areas -Cancer- in Ministry of Education, Science, Sports and Culture of Japan ("Cell cycle control of DNA replication" organized by Tsurimoto) during the period of 1996-1999. We would like to thank the financial support of our research activities by this funding and especially thank Professor Yoshiaki Ito of Kyoto University for his generous support of above research activity throughout the course of this project.