Universitą degli studi di Pavia


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Functional Dissection of a Human Replicator
DNA replication initiates by recruiting the replication machinery to thousands of sites scattered on chromosomes, termed replication initiation sites or replication origins. Origin selection is mediated by the assembly of the pre-replication complex, which initiates with the binding of the Origin Recognition Complex (ORC) onto replication origins. It is not yet fully understood whether the targeting of metazoan ORC to specific replication origins, relies on the presence of permissive local chromatin structure, DNA topology and/or accessory targeting factors that exhibit sequence-specificity. Although the metazoan ORC exhibits virtually no sequence-specificity and metazoan origins do not share any consensus sequence, the replication initiation events are nevertheless not random, as determined by studies on several replicators including the origin associated with the human Lamin B2 gene (LamB2-ori). Replication origins may be active even upon integration into ectopic sites of the host genome, supporting a genetic process of origin selection. Indeed, we previously cloned a 1.2kb DNA fragment of the LamB2-ori (eLamB2-ori), expanded the plasmid vector in bacteria to erase any epigenetic code, randomly integrated the ectopic origins in the host genome and showed that the eLamB2-ori was still active. We applied Recombinase Mediated Cassette Exchange (RMCE) to integrate several mutants of the eLamB2-ori in a constant genomic site to minimize the positional effects of flanking sequences. By comparing the firing activity of LamB2-ori mutants, we intend to define the minimal sequence of the replicator and identify the sequence elements required for triggering initiation of replication. The composition of the complex interacting with the origin mutants is under investigation by chromatin immunoprecipitation assay. Moreover, we are also investigating the interplay between epigenetic code and origin firing as well as understanding the role of chromatin binding factors in the processes of origin selection and timing of activation.

Defining the Sequence Elements of the Human LamB2-ori Replicator Involved in the Prevention of Gene Silencing
Depending on the stage of development, growth conditions, or cell transformation status, in mammals approximately 10’000-1’000'000 replication origins initiate DNA replication at variable frequency from well-defined sites or from multiple sites within a region. Origin density is strongly correlated with genomic landscapes, with clusters of closely spaced origins in GC-rich regions and no origins in large GC-poor regions. Several origins map within or near CpG islands and/or overlap to transcriptional regulatory elements suggesting a putative role in gene expression. It was also shown that origins of replication may prevent gene silencing. In this project, we have been investigating the role of the origin sequence elements in preventing gene silencing and identifying chromatin-binding proteins involved in this process.
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