DNA replication is a crucial step in the eukaryotic cell cycle. Activation of replication origins in early S phase requires the assembly of a pre-replication complex that is subsequently modulated into a pre-initiation complex and finally into a fully active initiation complex. This cascade of events is initiated in Gl phase of the cell cycle and terminates with the establishment of 2 bidirectionally moving, processive replication forks. The evolutionarily conserved protein Mcm10 has been implicated in both the initiation and elongation steps of DNA replication. However, Mcm10's precise role in these processes has remained elusive. Our studies on Mcm10 in S. cerevisiae suggest that Mcm10 is cell cycle regulated and an essential component of the replication fork. Furthermore, we have shown that Mcm10 controls the stability of the catalytic subunit of DNA polymerase-alpha. Our preliminary results suggest that Mcm10 also interacts with DNA polymerase-epsilon and PCNA (proliferating cell nuclear antigen). Thus, Mcm10 appears to be a key coordinator in replication fork assembly and is therefore, a putative target for anti-cancer treatment. The experiments proposed in this grant application are targeted to achieve a better understanding of Mcm10 and its interactions with the catalytic subunit of DNA polymerase-alpha and other replication proteins.
The specific aims of this proposal are: 1. What is Mcm10's role in initiation complex assembly? We hypothesize that in addition to regulating the stability of pol-alpha Mcm10 participates in preinitiation complex assembly. 2. What is Mcm10's role at replication forks? We hypothesize that Mcm10 interaction with PCNA is required for DNA elongation. 3. How does Mcm10 stabilize DNA polymerase-alpha? Our hypothesis is that specific domains in Mcm10 are required to stabilize Cdc17. 4. What is the mechanism of Cdc17 degradation in the absence of Mcm10? Loss of Mcm10 triggers the degradation of Cdc17. However, it remains unclear how Cdc17 is degraded. Cdc17 has 2 matches to PEST-like sequences, which have been implicated as signal sequences for ubiquitin-mediated proteolysis. Our working hypothesis is that binding to Mcm10 renders these PEST sequences inaccessible to the degradation machinery.
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