Human minichromosome maintenance proteins and human origin recognition complex 2 protein on chromatin.
Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.
Ritzi
M
M
Department, Universität Konstanz, D-78457 Konstanz, Federal Republic of Germany.
Baack
M
M
Musahl
C
C
Romanowski
P
P
Laskey
R A
RA
Knippers
R
R
D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000032:analysis / Q000378:metabolism*; D002467:Cell Nucleus / Q000502:physiology / Q000648:ultrastructure; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D051738:Origin Recognition Complex; D012097:Repressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D012097:Repressor Proteins; D008836:Micrococcal Nuclease