ABSTRACT
The protein stability of the initiation factors Orc2, Orc3, Orc4, and Cdc6 was analyzed after UV light exposure in two human cell lines. In the cell line with higher repair capacity, HEK 293, no changes in the cell cycle distribution or in the protein levels of the investigated factors were detected. In HeLa cells that are characterized by lower repair capacity, UV irradiation caused a reduction of the levels of Cdc6, Orc2 and Orc3, but not of Orc4 or triggered apoptosis. The appearance of the truncated 49 kDa form of Cdc6 suggested the involvement of the caspase pathway in the degradation of the proteins. Reduced protein levels of Cdc6 were detected in UV damaged HeLa cells in which the apoptotic process was blocked with the caspase inhibitor Z-VAD-fmk, indicating that the degradation of Cdc6 is mediated by the proteasome pathway instead. In the presence of caffeine, an inhibitor of the cell cycle checkpoint kinases, Cdc6 was stabilized, demonstrating that its degradation is controlled by the DNA damage cell cycle checkpoint. We conclude that in response to DNA damage, the activation of origins of replication can be prevented by the degradation of Cdc6, most likely through the proteasome pathway.
Subject(s)
Cell Cycle Proteins , Origin Recognition Complex , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Damage , DNA Replication , HEK293 Cells , HeLa Cells , Humans , Origin Recognition Complex/genetics , Origin Recognition Complex/metabolism , Protein Stability , Ultraviolet RaysABSTRACT
The proteins bound in vivo at the human lamin B2 DNA replication origin and their precise sites of binding were investigated along the cell cycle utilizing two novel procedures based on immunoprecipitation following UV irradiation with a pulsed laser light source. In G(1), the pre-replicative complex contains CDC6, MCM3, ORC1 and ORC2 proteins; of these, the post-replicative complex in S phase contains only ORC2; in M phase none of them are bound. The precise nucleotide of binding was identified for the two ORC and the CDC6 proteins near the start sites for leading-strand synthesis; the transition from the pre- to the post-replicative complex is accompanied by a 17 bp displacement of the ORC2 protein towards the start site.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Replication Origin , Ultraviolet Rays/adverse effects , Base Sequence , Cell Cycle , Cell Cycle Proteins/genetics , Cross-Linking Reagents/pharmacology , DNA/chemistry , DNA/metabolism , DNA Replication/drug effects , DNA Replication/radiation effects , DNA-Binding Proteins/chemistry , HeLa Cells , Humans , Lamin Type B/metabolism , Lasers , Minichromosome Maintenance Complex Component 3 , Nuclear Proteins/genetics , Origin Recognition Complex , S Phase , Time FactorsABSTRACT
HeLa cells were synchronized at late G1, early S, and late S phase of the cell cycle by nocodazole treatment. The cells were permeabilized with Triton X-100, digested with DNAse I, and extracted with 0.2 M ammonium sulfate to remove the digested chromatin. DNA was isolated from the residual chromatin attached to the nuclear matrix, digested with Hind III, and subjected to hybridization with [(32)P] labeled probe located upstream of the core region of the human beta-globin replication origin. The hybridization pattern revealed the existence of a DNase I sensitive site in the core region of the beta-globin replicator. The results suggest that association with the nuclear matrix induce alteration in the chromatin structure of the origin of replication that represents a more open chromatin configuration.
