Modular structure of the human lamin B2 replicator |
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Authors: | Paixão Sónia Colaluca Ivan N Cubells Matthieu Peverali Fiorenzo A Destro Annarita Giadrossi Sara Giacca Mauro Falaschi Arturo Riva Silvano Biamonti Giuseppe |
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Institution: | Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy. |
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Abstract: | The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation. |
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