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Mol. Cell. Biol. doi:10.1128/MCB.02382-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The role of Orc6 protein in ORC-dependent DNA binding and replication in Drosophila

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, 720 20th Street South, Birmingham, AL 35294

^* To whom correspondence should be addressed. Email: ichesnokov{at}uab.edu.

	Abstract

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of Orc6 protein that does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.

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