Tesi Finale Sannino.pdf

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Tipologia del documento: Tesi di dottorato
Lingua: English
Data: 25 Marzo 2013
Numero di pagine: 99
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Chimica organica e biochimica
Scuola di dottorato: Biotecnologie
Dottorato: Scienze biotecnologiche
Ciclo di dottorato: 25
Coordinatore del Corso di dottorato:
Rossi, Mosè
Pisani, Francesca
Data: 25 Marzo 2013
Numero di pagine: 99
Parole chiave: Cdc45, DNA replication, CMG complex, MCM2-7 complex, GINS complex
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Depositato il: 03 Apr 2013 14:35
Ultima modifica: 23 Lug 2014 09:40
DOI: 10.6092/UNINA/FEDOA/9102


DNA replication is a crucial step for cell survival. All the information useful for the cells to propagate and complete their tasks is indeed kept in the DNA molecules that are needed to be duplicated in a proper way. Cdc45 is a fundamental factor involved both in the initiation and in the elongation phases of the DNA replication process. According to its function in the DNA replication mechanism it has been shown to be a proliferation-associated antigen and in fact it is expressed only in proliferating cells. Even if a peak for the mRNA codifying for Cdc45 can be observed at the G1/S border, levels of Cdc45 are stable throughout the cell cycle in proliferating cells. The loading of Cdc45 onto the chromatin has been shown to coincide with the origin firing (i.e. replication origins activation). During late M and G1 phases of the cell cycle, replication origins, the peculiar sequences on the genome where DNA replication process is supposed to start, are bound by the Origin Recognition Complex (ORC). This latter is itself bound by two other factors, Cdc6 and Cdt1, which are involved in the loading onto the chromatin of the MCM2-7 complex. This complex represents the molecular motor of the replicative DNA helicase, but at this stage it remains in an inactive state. At the G1/S border the GINS complex and Cdc45 are recruited at the replicative forks and associate with the MCM2-7 complex, bringing to the formation of the so called CMG complex, which is finally able to start working as DNA helicase, unwinding the DNA helix which can now be duplicated by the DNA polymerases. Even if the peculiar mechanism by which Cdc45 is loaded onto DNA is not fully understood, it is known that the activity of CDK2 and DDK kinases is required to this purpose. Cdc45 is the last factor to be loaded onto chromatin at the replicative fork and it is actually used by cells to switch on the DNA replication process. Once DNA replication has started, Cdc45 will keep on moving with the replicative fork co-operating together with GINS and MCM2-7 to provide DNA helicase activity. When we started our analyses on the human Cdc45 factor, not much was known about the biochemistry of this protein. Indeed, no structural data were available for Cdc45 and no enzymatic activity was known to be associated with it. We carried out a Blast search, using the human Cdc45 aminoacidic sequence as query, looking into archaeal genome databases. We started from the observation that all the other factors forming the eukaryotic CMG complex were found to have a counterpart in the archaeal DNA replication machinery, while no homologous was known for Cdc45 in these organisms. The result we got was actually highlighting a match between the human Cdc45 protein and an archaeal protein belonging to the DHH protein superfamily, which includes a wide range of phosphoesterases present in all organisms (i.e. Bacteria, Archaea and Eukarya). Comparing the human Cdc45 primary sequence with the ones from many other bacterial and archaeal DHH super-family members, a good degree of similarity can be observed in the N-terminal portion of these sequences. Comparison of the secondary structure elements of the core of DHH proteins with the predicted secondary structure motifs for the human Cdc45 protein, a good degree of similarity can be observed along the whole sequences. We produced and purified human Cdc45 from E. coli cells in recombinant form and we carried out SAXS analyses on this protein, in collaboration with Dr. Silvia Onesti (Sincrotrone, Trieste). The reconstruction model based on the results of these analyses shown that human Cdc45 seems to be folded in a kind of cornet shape, with a more compact central core and two long lateral extensions. The crystal structure of the Thermus thermophilus RecJ-like protein (a DHH phopshoesterase) can be properly superimposed with the central compact core of the hCdc45 predicted structure. Instead the two lateral extensions can be attributed to two long aminoacidic insertions which are not present in the bacterial proteins. Since DHH phosphoesterases usually bind two metal ions that are required for their catalytic activity, we checked if also the human Cdc45 protein was able to bind any kind of metal ion. According to the absence of most of the aminoacidic residues responsible for binding metal ions in the DHH proteins, Cdc45 was found not to bind any metal ion and, in agreement with this finding, it was found to show neither phyrophosphatase nor DNA exonuclease activity. We demonstrated that human Cdc45 has the ability to bind single-stranded DNA, while it shows no affinity for double-stranded DNA or RNA molecules. All these results were in agreement with the idea that Cdc45 has evolved from an ancestor DHH protein, that lost the enzymatic activity and just retained the ability to bind single-stranded DNA. We demonstrated that the N-terminal region of Cdc45 is not involved in the DNA binding activity, which can be likely ascribed to the C-terminal portion of the protein. Moreover, by using the Xenopus laevis egg extract experimental system we demonstrated that the “L1 loop”, a long aminoacidic insertion which is unique to the eukaryal Cdc45 proteins and is absent in the related archaeal and bacterial proteins, is not needed for the role played by Cdc45 in DNA replication in not perturbed conditions. To study the role played by Cdc45 in association with GINS and MCM2-7, we finally set up a method to produce the CMG complex in insect cells co-infected with multigenic baculoviruses.

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