Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40-infected monkey kidney cells.
BACKGROUND
Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin.


METHODS
Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content.


RESULTS
MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase.


CONCLUSIONS
The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.
Friedrich
Thomas D
TD
Albany Medical College, Center for Immunology and Microbial Diseases, Albany, New York 12208, USA. friedrt@mail.amc.edu
Bedner
Elzbieta
E
Darzynkiewicz
Zbigniew
Z
Lehman
John M
JM
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000378:metabolism; D015153:Blotting, Western; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005057:Eukaryotic Cells / Q000378:metabolism / Q000821:virology; D007150:Immunohistochemistry; D047569:Laser Scanning Cytometry; D008856:Microscopy, Fluorescence; D064111:Minichromosome Maintenance Complex Component 2; D015530:Nuclear Matrix / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D016196:S Phase / Q000502:physiology*; D013539:Simian virus 40 / Q000254:growth & development / Q000276:immunology; D013997:Time Factors; D014779:Virus Replication / Q000502:physiology
D000957:Antigens, Viral, Tumor; D018797:Cell Cycle Proteins; D002843:Chromatin; D009687:Nuclear Proteins; D004247:DNA; D064111:Minichromosome Maintenance Complex Component 2