Stubbs Matthew C, Hall David J
Cartilage and Orthopaedic Branch, National Institutes of Arthritis and Musculoskeletal and Skin Diseases, NIH, MSC 5755, 9000 Rockville Pike, Bethesda, Maryland, MD 20892, USA.
Oncogene. 2002 May 23;21(23):3715-26. doi: 10.1038/sj.onc.1205473.
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.
E2F1转录因子在驱动细胞脱离静止状态并进入细胞周期的S期过程中发挥着关键作用，部分原因是它能反式激活DNA复制所需的基因，包括二氢叶酸还原酶（DHFR）、胸苷激酶和DNA聚合酶α。E2F1也被认为参与调节S期检查点，然而其在该检查点中的作用尚未明确界定。为了确定E2F1如何影响这样一个检查点，我们利用了一种体内复制测定法，该方法采用基于质粒的SV40复制起点，将其转染到表达SV40大T抗原的细胞中。在这里我们表明，全长E2F1的表达，或仅其N末端的表达，会抑制含有SV40起点的质粒的复制，而E2F1的N末端缺失则不会。E2F1似乎抑制复制的延伸阶段而非起始阶段，因为它不影响其他仅含有SV40起点的共转染质粒的复制。此外，复制的抑制既依赖于E2F1蛋白的氨基末端，也依赖于E2F1 cDNA 3'端所含的一个DNA序列。另外，全长E2F1或仅其N末端，都会与E2F1 cDNA 3'端的两个部分形成蛋白质复合物。这些数据为E2F1通过其氨基末端区域作用于特定DNA序列来抑制DNA复制延伸从而调节通过S期检查点的机制提供了线索。
