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. Method: 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. (c) 2005 international society for Analytical Cytology.