n previous studies of DNA replication in drosophila ovary follicle cells we discovered a new complex thai regulates both gene expression and DNA replication in many cell types. This MMB/Dream complex remains one major focus for continued study for us during the next period of this project. The complex contains multiple DNA binding activities including an MJ hook in the core protein Mip 130 and specific DNA binding functions in Mip 120, Myb and E2f2/DP/Rb proteins. We propose experiments to explore how this complex may work to either repress or activate proximal origins of DNA replication or promoters for gene expression in one cell type and in a developmental setting switch signs for activity at discrete locations. How the different DNA binding factors touch DNA at different sites will be explored. One over-arching hypothesis to be tested is that post-translational protein modifications (PTMs) mediated.by signalling kinases, other proximal site-specific DNA factors and MMB composition together sen/e as ancf/or logic gates for function. Another hypothesis to be tested is that the precise structure and composition of the MMB nucleoprotein complex at discrete sites is critical for function. Furthermore we will test the notion that ancillary factors that recognize histone modifications are targeted to specific loci by the MMB and that histone tail interactions are critical for the decision for either repression or activation of a replication ori or a promoter. Integration of chromatin structure as mediated by complexes such as the MMB/Dream with the recruitment of proteins required for the basic function of a replication origin is another goal of this project. The MCM2-7 proteins ofthe eukaryote DNA replication machinery are recruited first as a pre-helicase to the pre-replication sites by concerted action of the six subunit ORC with CDC6 and Ctd-1. We will probe the structure and function of ORC together with Cdc6 and ask if such complexes may distort DNA for the loading of this inactive helicase. Activation of helicase activity requires the association of the GINS and Cdc 45 proteins to such pre-replication complexes creating the active CMG complex. We will study the structure and function ofthe CMG helicase and explore the roles of PTMs mediated by the Cdc7/Dbf4 kinase in linking the CMG to functional replisomes.

Public Health Relevance

The replication of DNA is a central step in the cell cycle and our work on the regulation and mechanisms of this process has uncovered'new protein complexes that are key factors widely conserved throughout multicellular animals including humans. Cancer and other proliferative diseases initiate with cellular events that transform and release normal cell cycle controls. The signaling events that control the action of replication proteins, will be provide insights into the networks that control cell cycle progression and should help in cancer therapy and diagnosis.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Daschner, Phillip J
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University of California Berkeley
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Petojevic, Tatjana; Pesavento, James J; Costa, Alessandro et al. (2015) Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement. Proc Natl Acad Sci U S A 112:E249-58
Bleichert, Franziska; Botchan, Michael R; Berger, James M (2015) Crystal structure of the eukaryotic origin recognition complex. Nature 519:321-6
Costa, Alessandro; Renault, Ludovic; Swuec, Paolo et al. (2014) DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome. Elife 3:e03273
Bell, Stephen D; Botchan, Michael R (2013) The minichromosome maintenance replicative helicase. Cold Spring Harb Perspect Biol 5:a012807
Bleichert, Franziska; Balasov, Maxim; Chesnokov, Igor et al. (2013) A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation. Elife 2:e00882
Ilves, Ivar; Tamberg, Nele; Botchan, Michael R (2012) Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. Proc Natl Acad Sci U S A 109:13163-70
Lewis, Peter W; Sahoo, Debashis; Geng, Cuiyun et al. (2012) Drosophila lin-52 acts in opposition to repressive components of the Myb-MuvB/dREAM complex. Mol Cell Biol 32:3218-27
Lyubimov, Artem Y; Costa, Alessandro; Bleichert, Franziska et al. (2012) ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote. Proc Natl Acad Sci U S A 109:11999-2004
Kawabata, Tsuyoshi; Luebben, Spencer W; Yamaguchi, Satoru et al. (2011) Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression. Mol Cell 41:543-53
Harrison, Melissa M; Li, Xiao-Yong; Kaplan, Tommy et al. (2011) Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet 7:e1002266

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