Structural and biochemical studies of MCM proteins

Medagli, Barbara (2010) Structural and biochemical studies of MCM proteins. [Tesi di dottorato] (Inedito)

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The eukaryotic MCM2-7 replicative helicase is a hetero-complex composed by 6 homologues belonging to the AAA+ ATPase superfamily. These proteins form a ring-like structure which is essential for the initiation and the progression of the replication fork. In order to better understand the cellular role and mechanism of action of these proteins, I carried out structural and functional studies on two different systems: the archaeal MCM proteins, that forms a homomeric ring representing a simplified model of the eukaryotic complex, and the less well studied human MCM8, whose exact role in eukaryotic cells has not been elucidated. MCM interacts in two ways with the DNA: the canonical "loaded" mode where the MCM protein is encircling the DNA, and a "associated" mode where the DNA is wrapped around the enzyme. Using the protein from the archaeon Methanothermobacter thermautotrophicum, I biochemically shown that binding of MCM proteins affect the degree of supercoiling of dsDNA and that subdomain A is crucial for this interaction. To determine the structure of the C-terminal domain of the MCM proteins, the less studied region, I produced three different constructs corresponding to the C terminus of the MCM proteins from M. thermoautotrophicum and Sulfolobus solfataricus. Despite a large effort using “high throughput” crystallisation techniques, no well diffracting crystals could be reproducibly obtained. An alternative structural biology approach has been adopted, using NMR analysis. Preliminary data are compatible with the bioinformatics prediction of a winged helix domain, but further work on isotope-labelled proteins is necessary for a full 3D characterisation. Two other MCM proteins have been more recently described (MCM8 and MCM9): they are present in a variety of eukaryotic organisms, do not interact with the MCM2-7 complex and their function in the cell is still unknown. These protein are co-evolutionary related and it is conceivable that they have a functional link. I cloned and expressed in E. coli cells a large number of hMCM8 fragments: two of them (the N-terminus and the AAA+ domain) can be successfully expressed in soluble form and have been purified and used for biochemical assays. Both proteins forms dimers, bind ssDNA and the AAA+ catalytic domain displays ATPase activity. Further effort will be done to optimise the purification protocol to obtain proteins suitable for structural studies. In order to assess the hypothesis of a functional relationship between MCM8 and MCM9 proteins, a similarly extensive cloning strategy was applied to human MCM9, with the aim of obtaining constructs expressing a variety of fragments, to be co-expressed with the equivalent hMCM8 domains, and to test the formation of functional complexes.

Tipologia di documento:Tesi di dottorato
Altre informazioni:tesi svolta presso: Biophysics Section, Department of Biological Sciences, Imperial College London Structural Biology laboratory, Sincrotrone Elettra, Trieste
Parole chiave:MCM helicase, DNA replication, AAA+
Settori scientifico-disciplinari MIUR:Area 05 Scienze biologiche > BIO/19 MICROBIOLOGIA GENERALE
Coordinatori della Scuola di dottorato:
Coordinatore del Corso di dottoratoe-mail (se nota)
Tutor della Scuola di dottorato:
Tutor del Corso di dottoratoe-mail (se nota)
De Felice,
Stato del full text:Accessibile
Data:30 Novembre 2010
Numero di pagine:133
Istituzione:Università degli Studi di Napoli Federico II
Dipartimento o Struttura:Biologia strutturale e funzionale
Tipo di tesi:Dottorato
Stato dell'Eprint:Inedito
Scuola di dottorato:Scienze biologiche
Denominazione del dottorato:Biologia applicata
Ciclo di dottorato:22
Numero di sistema:8211
Depositato il:02 Dicembre 2010 12:06
Ultima modifica:29 Agosto 2012 13:38

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