Research at the DNA replication group aims at understanding 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 [1]. Genetic dissection of replicators (for details, see Specific Aim 1) allows us to delineate the sequence requirements for starting DNA replication. We had also reported (Specific Aim 2) that the timing of DNA replication during the S-phase of the cell cycle can be altered [2]. We now use the ability to 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 evaluate the effect of changes in metabolic conditions and exposure to anti-cancer drugs on initiation patterns [3] (Specific Aim 3). The studies outlined 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. For this aim, we have established that the replication initiation region within the human _eta-globin locus contains two independent, non-overlapping replicators and have identified sequence motifs that are required for initiation of DNA replication within these replicators. [4] Although we have shown replicator sequences were not conserved during recent mammalian evolution [5], and that replication often requires interactions with distal sequences [6], replicators share common sequence features [7]. We reported that 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 [8]. Ongoing studies have begun to characterize proteins that bind critical sequences that affect the location of initiation events using a combination of biochemical techniques and genomic footprinting. 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 [2, 9] 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 [10] and histone modifications [2, 11]. Tissue-specific patterns of replication timing can be conserved in evolution even in loci that do not conserve replication initiation patterns [5]. We have shown that functional replicator sequences (but not mutated replicators) prevented gene silencing and replication delay and prohibited chromatin condensation [11]. Ongoing studies have analyzed in detail the role of certain DNA sequences that affect chromatin condensation, termed insulators, on the timing of DNA replication. 3. Identification of cellular signalling interactions induced by the perturbation of DNA replication. We have started to investigate how cells respond to perturbation of DNA replication. We have shown that exposure to mild drug-induced perturbation of DNA replication, which is below the threshold of the cell cycle checkpoint response, can rapidly induce DNA breaks. In cells that contain an intact nonhomologous end-joining pathway, those DNA breaks are transient and cells rapidly resume replication in the presence of the inhibitor, albeit at a slow rate. However, DNA breaks persist in cells that are deficient in components of the 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 [3]. Ongoing studies determine the role of other DNA-modifying proteins in the cellular response to perturbed replication. Cited references: 1. Aladjem, M.I., Rodewald, L.W., Kolman, J.L., and Wahl, G.M. (1998). Genetic dissection of a mammalian replicator in the human beta-globin locus. Science 281, 1005-1009. 2. Lin, C.M., Fu, H., Martinovsky, M., Bouhassira, E., and Aladjem, M.I. (2003). Dynamic alterations of replication timing in mammalian cells. Curr Biol 13, 1019-1028. 3. Shimura, T., Martin, M.M., Torres, M.J., Gu, C., Pluth, J.M., DiBernardi, M.A., McDonald, J.S., and Aladjem, M.I. (2007). DNA-PK is involved in repairing a transient surge of DNA breaks induced by deceleration of DNA replication. J Mol Biol 367, 665-680. 4. Wang, L., Lin, C.M., Brooks, S., Cimbora, D., Groudine, M., and Aladjem, M.I. (2004). The human beta-globin replication initiation region consists of two modular independent replicators. Mol Cell Biol 24, 3373-3386. 5. Aladjem, M.I., Rodewald, L.W., Lin, C.M., Bowman, S., Cimbora, D.M., Brody, L.L., Epner, E.M., Groudine, M., and Wahl, G.M. (2002). Replication initiation patterns in the beta-globin loci of totipotent and differentiated murine cells: evidence for multiple initiation regions. Mol Cell Biol 22, 442-452. 6. Aladjem, M.I., Groudine, M., Brody, L.L., Dieken, E.S., Fournier, R.E., Wahl, G.M., and Epner, E.M. (1995). Participation of the human beta-globin locus control region in initiation of DNA replication. Science 270, 815-819. 7. Aladjem, M.I., and Fanning, E. (2004). The replicon revisited: an old model learns new tricks in metazoan chromosomes. EMBO Rep 5, 686-691. 8. Wang, L., Lin, C.M., Lopreiato, J.O., and Aladjem, M.I. (2006). Cooperative sequence modules determine replication initiation sites at the human beta-globin locus. Hum Mol Genet 15, 2613-2622. 9. Feng, Y.Q., Warin, R., Li, T., Olivier, E., Besse, A., Lobell, A., Fu, H., Lin, C.M., Aladjem, M.I., and Bouhassira, E.E. (2005). The human beta-globin locus control region can silence as well as activate gene expression. Mol Cell Biol 25, 3864-3874. 10. Feng, Y.Q., Desprat, R., Fu, H., Olivier, E., Lin, C.M., Lobell, A., Gowda, S.N., Aladjem, M.I., and Bouhassira, E.E. (2006). DNA methylation supports intrinsic epigenetic memory in mammalian cells. PLoS Genet 2, e65. 11. Fu, H., Wang, L., Lin, C.M., Singhania, S., Bouhassira, E.E., and Aladjem, M.I. (2006). Preventing gene silencing with human replicators. Nat Biotechnol 24, 572-576
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