The Roles of the Mcm2-7 Tails in Replication Initiation
DNA replication is complex biological reaction that spans two stages of the eukaryotic cell cycle. In G1 phase, preparation for replication begins when two copies of the replicative helicase, the Mcm2-7 complex, are loaded onto DNA. However, these helicases are loaded in an inactive state, and are n...
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| Формат: | Диссертация |
| Опубликовано: |
Massachusetts Institute of Technology
2023
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| Online-ссылка: | https://hdl.handle.net/1721.1/147266 https://orcid.org/0000-0002-4893-8167 |
| Итог: | DNA replication is complex biological reaction that spans two stages of the eukaryotic cell cycle. In G1 phase, preparation for replication begins when two copies of the replicative helicase, the Mcm2-7 complex, are loaded onto DNA. However, these helicases are loaded in an inactive state, and are not activated until the following S phase. As cells enter S phase, the action of two kinases, DDK and S-CDK, facilitates the recruitment of two key helicase activators, Cdc45 and GINS. These proteins stably associate with Mcm2-7 to form the active replicative helicase complex called the CMG (Cdc45-Mcm2-7-GINS) complex. CMG complex formation is followed by DNA unwinding, replisome assembly, DNA synthesis, and finally replication termination. The Mcm2-7 helicase is involved in every step of this process, from the earliest stages of replication initiation to the final step of replication termination.
The Mcm2-7 helicase is a heterohexameric complex comprised of six related subunits. Each subunit contains an AAA+ ATPase domain, a large OB-fold domain, and extensions of varying lengths on each terminus. Importantly, Mcm2, Mcm4, and Mcm6 contain long unstructured N-terminal tails, which are unrelated to each other and whose role in replication initiation is not well understood.
In the following thesis, I describe the use of reconstituted helicase loading and helicase activation reactions to determine the specific contributions of the three Mcm2-7 N-terminal tails in key steps of replication initiation. Using this approach, I identified unique roles for each of the tails. First, I discovered that a 23 amino-acid region within the Mcm2 N-terminal tail is important for helicase loading. Second, I identified a role for the rest of the Mcm2 tail in facilitating the activation of the helicase by promoting the DDK phosphorylation of the Mcm4 and Mcm6 tails. Third, I found a unique role for the Mcm4 tail in maintaining DDK specificity of the helicase activation reaction thereby preventing unregulated replication initiation. Finally, I observed that the DDK phosphorylation sites on the Mcm4 and Mcm6 tails serve unique functions and cannot compensate for each other. |
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