Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster.
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 the Orc6 protein which 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.
Balasov
Maxim
M
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, School of Medicine, 720 20th Street South, Birmingham, AL 35294, USA.
Huijbregts
Richard P H
RP
Chesnokov
Igor
I
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001973:Bromodeoxyuridine; D002875:Chromosomes / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D004261:DNA Replication; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D051738:Origin Recognition Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011061:Poly A / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism
D000596:Amino Acids; D029721:Drosophila Proteins; D050505:Mutant Proteins; C477598:ORC6 protein, Drosophila; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D011061:Poly A; D004247:DNA; D001973:Bromodeoxyuridine