Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767847
Title: Mechanisms and regulation of dsDNA break repair in the Sulfolobus genus of thermophilic archaea
Author: Bray, Sian Marian
ISNI:       0000 0004 7651 2774
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Abstract:
DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.
Supervisor: Robinson, Nicholas P. Sponsor: BBSRC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.767847  DOI:
Keywords: Archaea ; DNA repair ; HerA ; NurA ; Mre11 ; Rad50 ; 20S proteasome ; Urm1 ; urmylation ; Thermophile ; Thermophilic Archaea ; Mre11/Rad50 ; Helicase ; Nuclease
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