Marson, Giuseppe
DNA, variations on the theme [Tesi di dottorato]

Molecular biology's central dogma, laid down in 1950s, affirmed that genetic information flows from DNA to RNA to protein synthesis. For a long time, the first element of this logic scheme, DNA, was regarded as inactive molecule with the sole purpose to act as a repository of genetic code. Indeed, the molecular dialogue between DNA and proteins has been generally interpreted as an univocal relationship between an inert partner (nucleic acid) and a versatile one (proteins), that remodels DNA as a clay object. Rather, has been emerged that each kind of interactions between macromolecules requires a mutual structural adaptation and chemical complementarity. As in Kurosawa’s classic movie “Rashomon” (1950) the same central event, a heinous crime, is recalled from the differing perspective of each character (a bandit, a samurai, samurai’s wife and a woodcutter) this present study aims to highlight some different, but complementary, aspects of the full dynamic repertoire of DNA macromolecules. In the first part of this thesis, I will demonstrate that DNA, according to a peculiar three dimensional arrangement, is not only a simple recipe for proteins production but something more. In particular, I will focus on guanosine quadruplex structure, a not canonical B-form of DNA could be described through multiple points of view. On one side, synthetic guanosine quadruplex can act as “smart” biomolecules able to recognize multiple targets, with potential implications both as diagnostic as well as therapeutic agent. This short DNA/RNA sequences, called aptamers, according to a unique molecular flexibility, are able to recognize and bind a broad range of targets with specificities reminiscent those exhibited by antibodies. As working model, here I will present a detailed characterization in vitro of not physiological guanosine rich sequences able to bind human thrombin, a protein of physiological and pathological relevance. Our research was aimed to describe the relative role of the structural modules composing their molecular architecture. This allowed us to propose a structure activity relationship of synthetic G quadruplex aptamers, in order to fully rationalize and optimize their binding property. On the other side, G-quadruplex forming sequences are also found in human genome. Some of them have been described as unique biochemical on/off switch able to regulate tumorigenic pathways. In particular, the expression of the oncogene c-Myc is controlled through the formation of non–B-form DNA structures within its promoter. The conformational shift of this promoter between a transcription inactive form (G-quadruplex form) to an active one (a canonical double strand form) is strictly regulated by several nuclear proteins. In the second section I’ll present a study concerning the heterologous expression, the purification scheme of the resulting products and the biochemical characterization of the functional domains of human nucleolin, a nucleolar protein that is able to inhibit c-Myc oncogene transcription by a peculiar recognition of its promoter in a G quadruplex form. This approach was pursued to deeper clarify the mechanism of this binding event. Doubtless, this represents a promising goal in order to develop new selective and effective chemotherapy drugs. Although revolutionary, the idea that genetic information was encoded only by DNA sequence, in a protein-free mechanism has been appeared definitely too simplistic. Indeed, in organisms with nuclei chromosomal DNA is organized along with protein templates (histones), forming a complex called chromatin. This is target of diverse array of posttranslational modifications that modulate the interaction among chromatin-associated proteins, which ultimately dictate dynamic transitions between transcriptionally active (euchromatin) or transcriptionally silent chromatin states (heterochromatin). In the last section, I will focus on the structural insights standing on the recognition event between a modified histone N-terminal tail and a specialized ‘effector’ protein (ORC1b), generally known for its role in pre-replication complex assembly. The identification of the molecular details that clarify how distinct protein modules are able to recognize specific histone modifications is a critical step to understanding how chromatin dynamics influence fundamental DNA-templated processes such as transcription, DNA recombination and DNA repair. In particular, our results identify the tandem PHD-BAH domains of Arabidopsis thaliana ORC1b as a novel unmethylated-lysine-binding module, thereby establishing the first direct link between histone methylation grade and the epigenetic role of ORC1b, previously known as a transcriptional regulation factor only for a series of specific interactions with silencing regulators.

In relazione con
CHIM/08 - Chimica farmaceutica

Tesi di dottorato. | Lingua: | Paese: | BID: TD16054143