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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37CA030490-32
Application #
8288597
Study Section
Special Emphasis Panel (NSS)
Program Officer
Daschner, Phillip J
Project Start
1981-07-01
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
32
Fiscal Year
2012
Total Cost
$650,681
Indirect Cost
$225,330
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
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
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
Costa, Alessandro; Ilves, Ivar; Tamberg, Nele et al. (2011) The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol 18:471-7
Ilves, Ivar; Petojevic, Tatjana; Pesavento, James J et al. (2010) Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell 37:247-58
Botchan, Michael; Berger, James (2010) DNA replication: making two forks from one prereplication complex. Mol Cell 40:860-1
Harrison, Melissa M; Botchan, Michael R; Cline, Thomas W (2010) Grainyhead and Zelda compete for binding to the promoters of the earliest-expressed Drosophila genes. Dev Biol 345:248-55
Clarey, Megan G; Botchan, Michael; Nogales, Eva (2008) Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping. J Struct Biol 164:241-9
Georlette, Daphne; Ahn, Soyeon; MacAlpine, David M et al. (2007) Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb MuvB/dREAM complex in proliferating cells. Genes Dev 21:2880-96
Beall, Eileen L; Lewis, Peter W; Bell, Maren et al. (2007) Discovery of tMAC: a Drosophila testis-specific meiotic arrest complex paralogous to Myb-Muv B. Genes Dev 21:904-19

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