The DNA Replication Group aims to understand how information from the cell cycle machinery leads to the initiation of DNA replication. Proper cell growth depends on a network of interacting molecules that prevents DNA replication and cell division under unfavorable conditions. Disruptions in the intricate balance between components of this network may lead to cancer;however, interfering with signals transmitted by the cell cycle signaling network is an important tool for cancer therapy. A better understanding of the cell cycle is fundamental to the development of rational, knowledge-based strategies to combat cancer and utilize stem cells to improve human health. To study cell cycle signaling at the chromatin level, we specify DNA sequences that determine whether, where, and when replication will occur. DNA sequences that determine the location of replication initiation are called replicators. Replicators are identified by their ability to start replication when transferred from their original genomic locus to ectopic genomic sites [Aladjem, MI, et al. Science 281: 1005-9, 1998]. Genetic dissection of replicators (see Specific Aim 1) delineates the sequence requirements for starting DNA replication. This year we have started a detailed analysis of DNA-protein interactions of these sequences. We also report (Specific Aim 2) that the timing of DNA replication during the S-phase of the cell cycle can be altered [Lin CM, et al. Curr Biol 13: 1019-28, 2003]. We now alter replication timing as a tool to elucidate genetic and epigenetic factors that determine replication timing. We have recently started to use single molecule analyses of DNA replication to determine replication timing of particular sequences (Specific Aim 2) and to evaluate the effect of changes in metabolic conditions and exposure to anti-cancer drugs on initiation patterns [Shimura T, et al. J Mol Biol 367: 665-80, 2007;375:1152-64m 2008] (Specific Aim 3). The studies outlined above provide insights into the interactions of the cell cycle machinery with chromatin to control DNA replication during normal growth and in response to replication-perturbing drugs. Below is a summary of recent findings, summarized briefly for each specific aim. 1) Characterization of replicators, genetic elements that affect the location of replication initiation. We have established that the replication initiation region within the human beta-globin locus contains two independent, non-overlapping replicators and have identified sequence motifs that are required for initiation of DNA replication within these replicators [Wang L, et al. Mol Cell Biol 24, 3373-86, 2004]. At the beta-globin locus, those sequence motifs interact with each other to determine the location of replication initiation events, implying a modular structure for mammalian replicators [Wang L, et al. Hum Mol Genet 15: 2613-22, 2006]. The above studies are in line with the emerging understanding that replication patterns in metazoans are dynamically regulated by a combination of sequence and epigenetic modifications [Aladjem MI. Nat Rev Genet 8: 588-600, 2007]. Our recent observations suggest that replicators bind particular proteins at specific stages of the cell cycle. We have identified a protein complex that affects replication timing and gene silencing by binding to a specific sequence in a replicator. We have evidence suggesting that a second replicator binding complex directly affect the location of replication initiation events. We are currently involved in identification of the second protein complex and in studying the role of the two protein-DNA interactions in the early stages of DNA replication. 2) Analysis of the effect of DNA sequence and chromatin structure on replication timing. We have identified DNA sequences that affect the timing of DNA replication [Lin CM, et al. Curr Biol 13: 1019-28, 2003;Feng YQ, et al. Mol Cell Biol 25: 3864-74, 2007] and have shown that the timing of DNA replication correlates with the status of chromatin condensation and with epigenetic factors, such as methylation of CpG sequences [Feng YQ, et al. PLoS Genet 2, e65, 2006] and histone modifications [Lin CM, et al. Curr Biol 13: 1019-28, 2003;Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. Tissue-specific patterns of replication timing can be conserved in evolution even in loci that do not conserve replication initiation patterns [Aladjem MI, et al. Mol Cell Biol 22: 442-5, 2002]. We have shown that functional replicator sequences (but not mutated replicators) prevented gene silencing and replication delay and prohibited chromatin condensation [Fu H, et al. Nat Biotechnol 24: 572-6, 2006]. We found that a protein-DNA interaction affects DNA silencing and characterized the protein involved in that interaction. We hve shown that point mutations that prevent this interaction affect the timing of DNA replication and allow gene silencing. In addition, the protein-DNA interaction we have characterized plays a role in determining how DNA methylation affects transcriptional silencing. These studies are under review for publication. In addition, we continue to collaborate with gene therapy laboratories to evaluate the relevance of the effects of replicators on gene silencing in the development of gene therapy vectors. 3) Identification of cellular signaling interactions induced by the perturbation of DNA replication. We have uncovered a cellular response pathway involving BLM helicase, Mus81 nuclease, ATR kinase and the non-homologous recombination cascade. This pathway responds to perturbation of DNA replication following exposure to mild drug-induced perturbation of DNA replication, which is below the threshold of the cell cycle checkpoint response. Cells that are exposed to mild perturbation of DNA replication exhibit transient DNA breaks that are formed by Mus81 with the cooperation of BLM helicase and ATR kinase [Shimura T, et al. J Mol Biol 375: 1152-64, 2008]. In cells that contain an intact nonhomologous end-joining pathway, these DNA breaks are transient and cells rapidly resume replication in the presence of the inhibitor, albeit at a slow rate. DNA breaks persist in cells that are deficient in components of the nonhomologous end-joining pathway such as DNA-PK and XRCC4;such cells are unable to resume DNA replication and activate a cell cycle checkpoint response after a mild inhibition of DNA synthesis [Shimura T, et al. J Mol Biol 367: 665-80, 2007]. These recent findings propose that replication-induced DNA breaks do not always arise passively from polymerase collisions;breaks can also form as intermediates in the resolution of perturbed replication. Massively parallel sequencing studies of replication initiation events also suggest that cells that cannot create and process the breaks have a distinct DNA replication profile during normal growth.