The retinoblastoma tumor suppressor protein (RB) is a critical regulator of cell proliferation. RB binds to the E2F transcription factor and together repress transcription of S-phase genes, thereby controlling the G1-S cell cycle transition. It has become increasingly clear that RB also has a direct function in regulating DNA replication at sites of replication initiation. The evidence for this direct role comes from five observations: (1) RB localizes to replication foci in primary human cells and regulates progression through S-phase as well as the G1-S transition. (2) RB interacts with the MCM7 replication factor and can repress replication in a Xenopus cell-free and transcription-free system. (3) RB can be recruited to mammalian replication origins after DNA damage and prevents endoreduplication. (4) Drosophila RB (RBF) interacts with the replication initiation factor, the Origin Recognition Complex (ORC). (5) Mutants in Drosophila E2F and RB fail to limit replication initiation and mis-localize ORC in ovarian follicle cells. The mechanisms by which RB functions to control replication directly are not known. This proposal builds on our previous observations that Drosophila RB associates with ORC and that RB mutants mislocalize ORC and fail to limit ? DNA replication. We now have shown that the N-terminus of RBF (a.a. 151-330), that is conserved ? throughout RB family proteins, is critical for its interaction with ORC. This and recent reports that human RB localizes to replication initiation sites strongly suggests that RB control of replication initiation is an evolutionarily conserved function. Our central hypothesis is that RB regulates DNA replication by two distinct mechanisms: First, it represses transcription of S-phase specific genes by responding to antiproliferative and developmental cues; Second, RB associates with ORC at origins to inhibit replication initiation. There are two big picture questions we are asking here. (1) What are the mechanisms by which RB directly represses replication initiation at origins? (2) How are endo cycles and chorion amplification regulated by developmental signals thus coordinating development with RB regulated cell cycle events? Using Drosophila oogenesis as a model system, our proposed studies will give us new insights as to how the RB tumor suppressor protein in response to developmental cues regulates DNA replication. ? ?
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