The amino-terminus of the E2F-1 transcription factor inhibits DNA replication of autonomously replicating plasmids in mammalian cells.
The E2F1 transcription factor plays a pivotal role in driving cells out of a quiescent state and into the S phase of the cell cycle, in part by transactivating genes needed for DNA replication including DHFR, thymidine kinase, and DNA Polymerase alpha. E2F1 has also been implicated in regulating an S phase checkpoint, however its role in this checkpoint is not well defined. To determine how E2F1 affects such a checkpoint, we utilized an in vivo replication assay employing a plasmid based SV40 origin of replication, transfected into cells expressing SV40 large T antigen. Here we show that expression of full length E2F1, or only its N terminus, represses replication from plasmids containing the SV40 origin, while N terminal deletions of E2F1 do not. E2F1 appears to inhibit the elongation phase of replication and not the initiation phase since it does not affect the replication of other cotransfected plasmids containing only the SV40 origin. Further, inhibition of replication is dependent on both the amino-terminus of the E2F1 protein and on a DNA sequence that is contained within the 3' end of the E2F1 cDNA. Additionally, both full-length E2F1, or just its N-terminus, form protein complexes with two portions of the 3' end of the E2F1 cDNA. These data provide a clue to the mechanism by which E2F1 regulates transit through the S phase checkpoint, by acting on a specific DNA sequence via its amino-terminal region, to inhibit elongation of DNA replication.
Stubbs
Matthew C
MC
Cartilage and Orthopaedic Branch, National Institutes of Arthritis and Musculoskeletal and Skin Diseases, NIH, MSC 5755, 9000 Rockville Pike, Bethesda, Maryland, MD 20892, USA.
Hall
David J
DJ
D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D004261:DNA Replication; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D024202:Electrophoretic Mobility Shift Assay; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D014157:Transcription Factors