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|Title:||Expression and biochemical studies of the human MCM2-7 helicase||Authors:||Di Crescenzio, Patrizia||Supervisore/Tutore:||Onesti, Silvia||Issue Date:||28-Apr-2015||Publisher:||Università degli studi di Trieste||Abstract:||
The MCM helicase plays a key role in DNA replication, as it unwinds the double helix, providing a single stranded template for the DNA polymerases. MCM genes are found both in Eukaryotes and Archaea. Eukaryotic genomes present at least 6 different MCM genes, which share significant sequence similarity. The six different polypeptides encoded by MCM genes assemble into a heteroexamer, characterized by a toroidal conformation. Each protein comprises 3 domains: the N-terminal domain binds DNA and is essential for hexamerization; the C-terminal domain folds as a winged-helix motif and may have a regulatory role; the central AAA+ domain is the catalytic core of the helicase that couples the ATP hydrolysis with DNA unwinding. Since the eukaryotic MCM complex is difficult to produce in large amounts, most of the available structural information derive from studies on the archaeal model. For eukaryotic MCM proteins no crystal structure has been determined yet, while low resolution reconstructions by electron microscopy are available only for S. cerevisiae and D. melanogaster complexes.
The MCM proteins are present only in proliferating cells and are highly expressed in malignant cancer cells and pre-cancerous cells undergoing malignant transformation. Their expression has been compared with routinely used proliferation markers, showing that they are suitable candidates as biomarkers for cancer in clinical practice. Therefore, a detailed knowledge of their structure and function is a crucial pre-requisite for their potential role in cancer diagnosis and therapy.
Based on bioinformatics analysis and structural information from archaeal MCM proteins, fragments corresponding to the AAA+ domains and N-terminal domains of the human MCM helicase were designed and cloned separately into different expression vectors. Various expression strategies were explored and constructs were tested for protein expression under a variety of conditions.
AAA+ domains showed a severe problem of solubility: most of them precipitated during purification, aggregated or, in the best cases, precipitated after tag removal. The attempt to co express or co purify the subunits together didn’t improve the solubility of the proteins, also in presence of ATP analogues.
The N-terminal domains were soluble and expressed at high levels. The co purification of the six fragments led to the assembly of two different oligomeric states compatible with a double and a single hexamer. Both oligomers fit the biological role of the protein, expected to be loaded onto the double stranded DNA as double hexamer and to unwind as single hexamer. DNA binding experiments were carried out by fluorescence polarization: both assemblies are able to bind different DNA substrates (single strand, double strand, fork) with micromolar dissociation constants, consistent with the DNA binding affinities reported for the archaeal MCM complex. A preliminary biophysical characterization performed with different techniques (thermal shift analysis, circular dichroism, multi angle light scattering, small angle X-ray scattering) revealed that the putative single hexamer is properly folded but quite unstable, as it showed the tendency to disassemble into smaller oligomers. In contrast, the putative double hexamer showed a higher stability, but still a degree of inhomogeneity which didn’t allow a structural study of its architecture. Nevertheless, the high yield of our purification protocol represents a promising starting point for further optimization, allowing crystallography and other structural studies.
|Ciclo di dottorato:||XXVII Ciclo||metadata.dc.subject.classification:||SCUOLA DI DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE||Description:||
|Keywords:||MCM; biophysical characterization; expression; purification; DNA; binding||Type:||Doctoral||Language:||en||Settore scientifico-disciplinare:||BIO/11 BIOLOGIA MOLECOLARE||NBN:||urn:nbn:it:units-14065|
|Appears in Collections:||Scienze biologiche|
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