Differential binding of replication proteins across the human c-myc replicator.
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 G(1)-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 G(1)-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 G(1)-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.
Ghosh
Maloy
M
Department of Biochemistry and Molecular Biology, Wright State University, 3640 Colonel Glenn Highway, Dayton, Ohio 45435, USA.
Kemp
Michael
M
Liu
Guoqi
G
Ritzi
Marion
M
Schepers
Aloys
A
Leffak
Michael
M
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics* / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D016193:G1 Phase; D016259:Genes, myc; D006367:HeLa Cells; D006801:Humans; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D011485:Protein Binding; D012093:Replicon; D017384:Sequence Deletion
C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C500359:DTD1 protein, human; C066470:MCM3 protein, human; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D004247:DNA; C101624:MCM7 protein, human; D064126:Minichromosome Maintenance Complex Component 3; D064168:Minichromosome Maintenance Complex Component 7