GEMC1, a novel factor required for chromosomal DNA replication.
Doctoral thesis, UCL (University College London).
In eukaryotic cells DNA replication begins from multiple origins. During the process of initiation, the DNA replication fork is established at each origin. In lower eukaryotes many factors required for chromosomal DNA replication have been identified. However, the regulation of DNA replication in complex multi-cellular organisms is still poorly understood. In this thesis I report the identification of GEMC1 (GEMinin Coiled-coil containing protein 1), a novel vertebrate factor belonging to a new protein family required to initiate chromosomal DNA replication. GEMC1 contains a domain similar to Geminin, a fundamental regulator of DNA replication (McGarry and Kirschner, 1998). GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. I show that Xenopus GEMC1 (XlGEMC1) binds the checkpoint and replication factor TopBP1, which promotes XlGEMC1 binding to chromatin during pre-replication complex (pre-RC) assembly. Moreover, I demonstrate that XlGEMC1 directly interacts with the replication factors Cdc45 and Cdk2/CyclinE by which it is heavily phosphorylated. Phosphorylated XlGEMC1 stimulates initiation of DNA replication. Inhibition of XlGEMC1 function with XlGEMC1 neutralizing antibodies prevents DNA replication onset by blocking Cdc45 loading onto chromatin. Inhibition of XlGEMC1 expression by morpholino antisense oligos is lethal for embryonic development. Furthermore, down-regulation of mouse GEMC1 (mGEMC1) expression by siRNA (small interfering RNA) oligos prevents initiation of DNA replication in somatic vertebrate cells. Data presented in this thesis suggest that GEMC1 promotes initiation of chromosomal DNA replication in higher eukaryotes by mediating TopBP1 and Cdk2 dependent Cdc45 recruitment onto replication origins.
|Title:||GEMC1, a novel factor required for chromosomal DNA replication|
|Open access status:||An open access version is available from UCL Discovery|
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