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Molecular Characterization of Mth203 protein

Bochiwal, C (2011) Molecular Characterization of Mth203 protein. PhD thesis, University of York.

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Abstract

In contrast to the wealth of molecular and biochemical information available concerning eukaryotic and bacterial replication, less is known about the molecular basis of replication initiation in archaea. In general, the archaeal proteins are simplified versions of their eukaryotic counterparts. Therefore, they are potentially simple model systems to understand the conserved events in DNA replication and other processes. Methanothermobacter thermautotrophicus, a thermophilic archaeon has a circular genome and a single origin of replication. It’s replication initiation proteins MthCdc6-1 and MthCdc6-2 are homologues of the eukaryotic Cdc6 and ORC proteins. It also contains a single minichromosome maintenance protein (MthMCM) homologue which forms a hexameric complex and acts as a DNA helicase. What loads the archaeal MCM helicase and how DNA replication initiation is regulated is still unknown. Mth203, a putative RNA helicase in M. thermautotrophicus has been shown to interact with MthCdc6-1 in a yeast two-hybrid screen and His-tagged full-length protein pull-downs. Mth203 is a SF2 family helicase. The proteins of this superfamily perform a wide array of functions in DNA/RNA processing and often are a part of multi-protein complexes. The Mth203 homologue (Mmp0457) in Methanococcus maripaludis has been co-purified with one of the MCM proteins (McmA) in this organism. Thus, it is likely that this protein interacts with the replicative machinery. The aim of this study was to further characterize Mth203 structure and function. The results demonstrate that Mth203 is a dimeric protein and possesses NTP-dependent RNA helicase activity and RNA independent ATPase activity. Mth203 also inhibits MthMCM DNA helicase activity. Fluorescence anisotropy assays have shown that Mth203 binds non-specifically to short DNA sequences and specifically to long origin sequences. Mth203 was found to be expressed in periodic manner throughout the cell cycle and MALDI-TOF analysis has revealed that Mth203 interacts with MthCdc6-1 and ribosomal proteins in protein pull-down assays from whole cell extracts of M. thermautotrophicus. Further gene knock-out studies with Mth203 homologue (Mmp0457) in M. maripaludis have shown that the protein is probably essential for cell survival and mmp0457 overexpression resulted in larger cell size with less DNA content. It is not yet clear whether Mth203 is involved in DNA replication and its specific role in RNA metabolism remains unknown. However, these findings provide a valuable insight into the potential role of Mth203.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Depositing User: Unnamed user with email cb587@york.ac.uk
Date Deposited: 27 Feb 2012 15:51
Last Modified: 08 Aug 2013 08:47
URI: http://etheses.whiterose.ac.uk/id/eprint/1771

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