Mammalian MCM loading in late-G(1) coincides with Rb hyperphosphorylation and the transition to post-transcriptional control of progression into S-phase.
BACKGROUND
Control of the onset of DNA synthesis in mammalian cells requires the coordinated assembly and activation of the pre-Replication Complex. In order to understand the regulatory events controlling preRC dynamics, we have investigated how the timing of preRC assembly relates temporally to other biochemical events governing progress into S-phase.


RESULTS
In murine and Chinese hamster (CHO) cells released from quiescence, the loading of the replicative MCM helicase onto chromatin occurs in the final 3-4 hrs of G(1). Cdc45 and PCNA, both of which are required for G(1)-S transit, bind to chromatin at the G(1)-S transition or even earlier in G(1), when MCMs load. An RNA polymerase II inhibitor (DRB) was added to synchronized murine keratinocytes to show that they are no longer dependent on new mRNA synthesis 3-4 hrs prior to S-phase entry, which is also true for CHO and human cells. Further, CHO cells can progress into S-phase on time, and complete S-phase, under conditions where new mRNA synthesis is significantly compromised, and such mRNA suppression causes no adverse effects on preRC dynamics prior to, or during, S-phase progression. Even more intriguing, hyperphosphorylation of Rb coincides with the start of MCM loading and, paradoxically, with the time in late-G(1) when de novo mRNA synthesis is no longer rate limiting for progression into S-phase.


CONCLUSIONS
MCM, Cdc45, and PCNA loading, and the subsequent transit through G(1)-S, do not depend on concurrent new mRNA synthesis. These results indicate that mammalian cells pass through a distinct transition in late-G(1) at which time Rb becomes hyperphosphorylated and MCM loading commences, but that after this transition the control of MCM, Cdc45, and PCNA loading and the onset of DNA replication are regulated at the post-transcriptional level.
Program in Molecular Oncology, Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL, USA.
Piyali
P
Mukherjee
Thinh V
TV
Cao
Sherry L
SL
Winter
Mark G
MG
Alexandrow
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002843:Chromatin; D006224:Cricetinae; D003412:Cricetulus; D004261:DNA Replication; D050684:E2F Transcription Factors / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D026341:Minichromosome Maintenance 1 Protein / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase / Q000502:physiology*
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D050684:E2F Transcription Factors; D026341:Minichromosome Maintenance 1 Protein; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D016160:Retinoblastoma Protein