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Title: Identification and biochemical characterization of a lagging-strand specific replication sub-complex with the helicaseprimase- polymerase enzymes
Author: Rannou, Olivier
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2013
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DNA replication is the most fundamental process in all living species. Faithful replication and regulation are crucial for genome stability. Replisomes are protein assemblies that replicate DNA: they unwind double stranded DNA, synthesize RNA primers on the leading strand and lagging strand, and synthesize new DNA. The replisome of the low G + C content Gram-positive bacteria such as Bacillus subtilis contains two different DNA polymerases: a processive high-fidelity replicative polymerase PolC on the leading and lagging strand, and an error-prone DnaE on the lagging strand, which extends RNA primers before hand-off to Pole. We found that DnaE interacts with the helicase DnaCBs and the primase DnaG, and that these proteins form a complex in vitro. These interactions, which position DnaE in the lagging strand half of the replisome as expected from previous studies, are analysed in more detail here. We show that protein-protein interactions within binary complexes and ternary complexes formed by DnaE, DnaG and DnaCBs are functionally relevant. DnaE partially inhibits the helicase and the primase activities, while DnaCBs and DnaG stimulate each other's activities. Neither the helicase nor the primase had any effect on DnaE polymerase activity; but, they improved its fidelity, likely via allosteric effects induced by direct proteinprotein interactions that lower the efficiency of nucleotide mis-incorporation in the catalytic site of DnaE. These regulations within the replisome lagging strand subunit might participate in the synchronization of synthesis of the lagging-strand synthesis with the leading-strand, and improve the fidelity of lagging strand synthesis. This, and the possible roles of DnaE in other events related to DNA synthesis related events are discussed here.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available