Dean of Biological Sciences Professor of Molecular Therapeutics*
*Affiliate, Division of Genetics, Genomics and Development
The chromosomes of eukaryotic cells contain cis-acting elements important for gene expression, replication, folding and structure, segregation and recombination. Among these regulatory sites only those involved in gene expression are well studied. The arrival of the Cas/CRISPR system for creating site directed mutations in chromosomes should dramatically increase the rate of discovery in these areas.
In eukaryotes each chromosome has many sites that serve as initiators for DNA replication in dividing cells. For multicellular eukaryotes the utilization of these sites changes during the course of development. The program of activation is poorly understood in metazoans and is epigenetic where proteins that control chromatin access rather than direct sequence recognition by the general replication factors is a dominant theme. Replication stress occurs when key replication factors are limiting as a result of hypomorphic mutation or when cells in a quiescent niche are induced to enter a division cycle through defaults in cell proliferation controls. Such stress may lead to chromosome breaks and accelerating damage drives cancer progression.
We purified the DmORC -complex (Drosophila origin recognition complex) first from extracts and were able to reconstitute the activity with recombinant proteins. This complex is part of the machinery that marks chromosomal DNA as sites "to be initiated" for replication. The execution point for ORC function is in G1 where a “latent helicase” activity is wrapped around the duplex. Conversion of the latent helicase into an active unwinding machine requires the association of 5 other proteins to this pre-replication assembly. In cells this conversion is the key switch step for S phase and regulated by the S phase promoting kinases. (Botchan Nature 2007). We have called this active form of the helicase the “CMG” (Ilves et al Mol Cell). How the latent helicase is converted to the active enzyme and how the CMG unwinds duplex is presently a key lab project. In other studies we are exploring cell cycle check-point controls that stop the unwinding under stress conditions. Recombinant ORC is now the focus of high-resolution structural studies to understand how ORC executes it’s key function with other proteins to load the latent helicase.
Beall et al (Nature 2002) first showed that a Myb protein-complex was a key factor in regulating site-specific DNA replication and this complex is now known to regulate transcription and DNA replication in different cell types. The core factors of this complex in turn recruit chromatin modifying factors to either repress or activate chromosome function. How the histone binding factor L(3)MBT is targeted to promoters and replication sites through the auspices of this complex is also a research direction.
Bleichert F, Balasov M, Chesnokov I, Nogales E, Botchan MR, Berger JM. A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation. Elife. 2013;2:e00882.
Bell SD, Botchan MR. The minichromosome maintenance replicative helicase. Cold Spring Harb Perspect Biol. 2013 Nov 1;5(11):a012807.
Ilves I, Tamberg N, Botchan MR. Checkpoint kinase 2 (Chk2) inhibits the activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. Proc Natl Acad Sci U S A. 2012 Aug 14;109(33):13163-70.
Lyubimov AY, Costa A, Bleichert F, Botchan MR, Berger JM. ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote. Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):11999-2004.
Lewis PW, Sahoo D, Geng C, Bell M, Lipsick JS, Botchan MR. Drosophila lin-52 acts in opposition to repressive components of the Myb-MuvB/dREAM complex. Mol Cell Biol. 2012 Aug;32(16):3218-27.
Harrison MM, Li XY, Kaplan T, Botchan MR, Eisen MB. Zelda binding in the early Drosophila melanogaster embryo marks regions subsequently activated at the maternal-to-zygotic transition. PLoS Genet. 2011 Oct;7(10):e1002266.
Costa A, Ilves I, Tamberg N, Petojevic T, Nogales E, Botchan MR, Berger JM. The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol. 2011 Apr;18(4):471-7.
Kawabata T, Luebben SW, Yamaguchi S, Ilves I, Matise I, Buske T, Botchan MR, Shima N. Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression. Mol Cell. 2011 Mar 4;41(5):543-53.
Harrison MM, Botchan MR, Cline TW. Grainyhead and Zelda compete for binding to the promoters of the earliest-expressed Drosophila genes. Dev Biol. 2010 Sep 15;345(2):248-55.
Ilves I, Petojevic T, Pesavento JJ, Botchan MR. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell. 2010 Jan 29;37(2):247-58.
Georlette D., Ahn S., MacAlpine D.M., Cheung E, Lewis P.W., Beall E.L., Bell S.P., Speed T, Manak J.R. and Botchan M. Genomic profiling and expression studies reveal both positive and negative activities for the Drosophila Myb-MuvB/dREAM complex in proliferating cells. Genes Dev. 2007 Nov 15;21(22):2880-96.
Beall EL, Lewis PW, Bell M, Rocha M, Jones DL, Botchan MR Discovery of tMAC: a Drosophila testis-specific meiotic arrest complex paralogous to Myb-Muv B. Genes Dev. 2007 Apr 15;21(8):904-19.
Botchan, M. Cell Biology: a switch for S phase. Nature. 2007. 445: 272-274.
Abbate, E.A., Voitenleitner, C., and Botchan, M.R. Structure of the papillomavirus DNA-tethering complex E2:Brd4 and a peptide that ablates HPV chromosomal association. Mol. Cell. 2006 Dec 28;24(6):877-89.
Clarey, M.G., Erzberger, J.P., Grob, P., Leschziner, A.E., Berger, J.M., Nogales, E. and Botchan, M. Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nature Struct. & Mol. Biol. 2006 Aug;13(8):684-90.
Moyer, S.E., Lewis, P.W. and Botchan, M.R. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA. 2006 Jul 5;103(27):10236-41.
Remus, D., Blanchette, M., Rio, D.C. and Botchan, M.R. Cdk phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of drosophila ORC. J. Biochem. 2005 Dec 2;280(48):39740-51.
Bandura, J.L., Beall, E.L., Bell, M., Silver, H.R., Botchan, M.R. and Calvi, B.R humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila. Curr. Biol. 2005 Apr 26;15(8):755-9.
Lewis PW, Beall EL, Fleischer TC, Georlette D, Link AJ, Botchan MR. Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex. Genes Dev. 2004 Dec 1;18(23):2929-2940.
Abbate EA, Berger JM, Botchan MR. The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2. Genes Dev. 2004 Aug 15;18(16):1981-96.
Beall EL, Bell M, Georlette D, Botchan MR. Dm-myb mutant lethality in Drosophila is dependent upon mip130: positive and negative regulation of DNA replication. Genes Dev. 2004 Jul 15;18(14):1667-80.
Remus D, Beall EL, Botchan MR. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 2004 Feb 25;23(4):897-907
Chesnokov I, Chesnokova ON, Botchan M. A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activities. Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9150-5
Beall EL, Manak JR, Zhou S, Bell M, Lipsick JS, Botchan MR. Role for a Drosophila Myb-containing protein complex in site-specific DNA replication. Nature. 2002 Dec 19-26;420(6917):833-7
Voitenleitner C, Botchan M. E1 protein of bovine papillomavirus type 1 interferes with E2 protein-mediated tethering of the viral DNA to mitotic chromosomes. J Virol. 2002 Apr;76(7):3440-51.
Chesnokov I, Remus D, Botchan M. Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):11997-2002.
Pflumm MF, Botchan MR. Orc mutants arrest in metaphase with abnormally condensed chromosomes. Development. 2001 May;128(9):1697-707.
Parker LM, Harris S, Gossen M, Botchan MR. The bovine papillomavirus E2 transactivator is stimulated by the E1 initiator through the E2 activation domain. Virology. 2000 May 10;270(2):430-43.
Harris SF, Botchan MR. Crystal structure of the human papillomavirus type 18 E2 activation domain. Science. 1999 Jun 4;284(5420):1673-7
Chesnokov I, Gossen M, Remus D, Botchan M. Assembly of functionally active Drosophila origin recognition complex from recombinant proteins. Genes Dev. 1999 May 15;13(10):1289-96.
Fouts ET, Yu X, Egelman EH, Botchan MR. Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J Biol Chem. 1999 Feb 12;274(7):4447-58.
Lehman CW, Botchan MR. Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4338-43.
Lim DA, Gossen M, Lehman CW, Botchan MR. Competition for DNA binding sites between the short and long forms of E2 dimers underlies repression in bovine papillomavirus type 1 DNA replication control. J Virol. 1998 Mar;72(3):1931-40
Pak DT, Pflumm M, Chesnokov I, Huang DW, Kellum R, Marr J, Romanowski P, Botchan MR. Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell. 1997 Oct 31;91(3):311-23.
Photo credit: Mark Joseph Hanson of Mark Joseph Studio.
Last Updated 2014-02-10