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Molecular and Cellular Biology, July 2006, p. 5270-5283, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.02137-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Differential Binding of Replication Proteins across the Human c-myc Replicator

Maloy Ghosh,1,{dagger} Michael Kemp,1,{ddagger} Guoqi Liu,1,{ddagger} Marion Ritzi,2 Aloys Schepers,3 and Michael Leffak1*

Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435,1 Institut für Mikrotechnik Mainz GmbH, Fluidics, Mathematical Modeling and Sensorics Department, Carl-Zeiss-Straße 18-20, D-55129 Mainz, Germany,2 GSF-Haematologikum, Department of Gene Vectors, Marchioninistr. 25, D-81377 Munich, Germany3

Received 3 November 2005/ Returned for modification 8 December 2005/ Accepted 27 April 2006

The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G1-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G1-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G1-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Wright State University, 3640 Colonel Glenn Highway, Dayton, Ohio 45435. Phone: (937) 775-3125. Fax: (937) 775-3730. E-mail: Michael.Leffak{at}wright.edu.

{dagger} Present address: Terry Fox Laboratory, British Columbia Cancer Agency, 675 West 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada.

{ddagger} These authors contributed equally to this work.

Molecular and Cellular Biology, July 2006, p. 5270-5283, Vol. 26, No. 14
0270-7306/06/$08.00+0     doi:10.1128/MCB.02137-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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