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Title: Biochemical characterization of MCM helicase activation in vitro
Author: On, K. F.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Eukaryotic DNA replication is highly regulated so that the whole genome is duplicated completely and exactly once during each cell cycle. This is achieved by tight temporal control over origin licensing and origin firing, the two distinct steps essential for replication initiation. During origin licensing, the eukaryotic replicative helicase Mcm2-7 complex is assembled onto replication origins in a process known as pre- Replicative Complex (pre-RC) assembly, which involves the pre-RC factors ORC, Cdc6 and Cdt1. The products of pre-RC assembly, head-to-head associated MCM double hexamers, are activated to unwind duplex DNA in a process called origin firing. During origin firing, Cdc45, the hetero-tetrameric GINS complex and other firing factors interact and remodel the MCM complex, leading to pre-Initiation Complex (pre- IC) formation. Consequently, MCM helicase activation and assembly of the Replisome Progression Complex (RPC) occur, leading to replication initiation. Activation of the MCM helicase involves a list of firing factors including the kinases cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and other factors such as Sld2, Sld3, Sld7, Dpb11, DNA polymerase ε and Mcm10. The mechanisms behind MCM activation are currently unknown. Reconstitution of replication initiation in vitro is necessary to provide mechanistic understanding of origin firing. However, whether there are unidentified factors involved in this step remains an open question. The objective of the current study was to identify novel factors potentially important for origin firing by partially reconstituting MCM helicase activation in vitro with several purified proteins and an S-phase whole-cell extract. In this study, I have successfully activated reconstituted pre-RCs with purified DDK and an S-phase whole-cell extract. MCM helicase activation was monitored by DNA replication in a cell-free replication system. The RPC components Cdc45 and GINS as well as other firing factors were specifically recruited to pre-RCs in a DDK- dependent manner, indicating that pre-IC assembly has occurred under this condition. Importantly, semi-conservative DNA replication in vitro was detected, suggesting that the reconstituted pre-RCs were competent for activation and are the precursor for DNA replication. This observation also indicated that the DDK phosphorylation step during origin firing has been reconstituted in a functional manner, leading to RPC formation and replication initiation. Full-length, covalently closed replication products were generated in our replication system in which soluble DNA templates were used. Intriguingly, replication could be origin-independent, suggesting that replication might not initiate from specific sites on the DNA template. Using mass spectrometry analysis, I identified interesting candidate proteins potentially important in regulating origin firing. In conclusion, the biochemical system reported in the current study provides an excellent tool to study various aspects of DNA replication in vitro. Several novel factors potentially important in regulating DNA replication initiation were also identified in this study. The mechanistic roles of these factors in origin firing will be interesting questions for future studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available