Direct regulation of DNA replication initiation by the retinoblastoma protein

The Retinoblastoma tumor suppressor protein (pRB) is a key negative regulator of the cell cycle and one of the first tumor suppressors to be cloned. Functional inactivation of pRB is linked with initiation and progression of several human cancers. Past studies in mammalian cells have suggested that pRB's growth inhibition and tumor suppressive functions are largely mediated by its role as a transcriptional regulator, the most significant being its function at the restriction point repressing E2F mediated transcription of replication proteins that are required for transition from G1 to S phase. However recent studies suggest that pRB may play an important role in regulating DNA replication by directly binding to and inhibiting components of the pre-replication machinery, but the underlying molecular mechanism remains to be characterized. In mammalian cells it is difficult to distinguish between transcription regulation by pRB/E2F and direct effects of pRB on DNA replication. But the effect of pRB on the step wise assembly of pre-replication proteins and the events of replication initiation can be studied in egg extracts in the absence of translation, by the addition of the translation inhibitor cycloheximide. Also, the replication proteins that are normally controlled by the E2F pathway are already present in the eggs to efficiently carry out a single round of DNA replication, thus any repression of E2F transcription by pXRB is functionally insignificant in this system. Hence we have chosen to study the Xenopus full-length pRB protein (pXRB) in the homogenous Xenopus egg extract as a model system, in order to further characterize this novel function of pRB in directly regulating DNA replication initiation in the absence of transcription mediated effects.;We have purified the recombinant full-length pXRB to near homogeneity and our results indicate that pXRB inhibits DNA replication in Xenopus egg extracts in the absence of transcription mediated effects. By further characterization of pXRB, we show that pXRB can be phosphorylated in a CDK dependent manner, and interact with Xenopus E2F and Mcm7 proteins and inhibit DNA replication initiation events. pXRB does not interfere with the recruitment of the pre-replication complex (pre-RC) proteins to chromatin, but inhibits DNA replication initiation by preventing the chromatin recruitment of Mcm10, a replication protein that helps in the activation of the pre-replication complex to the pre-initiation complex (pre-IC), and thereby inhibiting pre-IC formation. By further analyzing a mutant of pXRB that can bind to Mcm7, but is compromised in binding to E2F, we show that direct inhibition of DNA replication by pXRB in the egg extracts does not require binding to E2F.;Our results also suggest that direct inhibition of DNA replication by pXRB may not be mediated exclusively through its binding to Mcm7 and that the sequences in both the amino terminus as well as carboxyl terminus of pXRB are required for DNA replication inhibition in the egg extracts. We also show that both Cyclin D as well as Cyclin E mediated phosphorylation of pXRB negatively regulates pXRB binding to chromatin, and relieves pXRB mediated inhibition of DNA replication initiation events. Finally in order to validate the results obtained with pXRB we also show that full-length human pRB can inhibit DNA replication in the egg extracts.;Taken together these results suggest a model in which pRB directly inhibits DNA replication initiation by binding to the pre-replication complexes on replication origins possibly through interactions with multiple pre-RC proteins in a functional hypophosphorylated state, and inhibit their activation by preventing downstream initiation events like Mcm10 recruitment and pre-IC formation. Upon mitogenic stimulation, CDK mediated phosphorylation of pRB then relieves this inhibition and allows for pre-RC activation and progression through S phase. Thus our studies point to another mechanism of regulation of replication licensing which is mediated by pRB, that efficiently links the Restriction point to temporal events of replication initiation. Hence, in addition to functioning indirectly though regulation of E2F at the restriction point, pRB might be part of a replication licensing checkpoint, ensuring the timely formation of the pre-initiation complex and origin activation, coupling mitogenic growth signals directly to replication initiation.