title: Modular structure of the human lamin B2 replicator.
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.
authors.affiliation: Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy.
authors.forename: Sónia
authors.initials: S
authors.lastname: Paixão
authors.forename: Ivan N
authors.initials: IN
authors.lastname: Colaluca
authors.forename: Matthieu
authors.initials: M
authors.lastname: Cubells
authors.forename: Fiorenzo A
authors.initials: FA
authors.lastname: Peverali
authors.forename: Annarita
authors.initials: A
authors.lastname: Destro
authors.forename: Sara
authors.initials: S
authors.lastname: Giadrossi
authors.forename: Mauro
authors.initials: M
authors.lastname: Giacca
authors.forename: Arturo
authors.initials: A
authors.lastname: Falaschi
authors.forename: Silvano
authors.initials: S
authors.lastname: Riva
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Biamonti
mesh_terms: D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D034921:Lamin Type B / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D051738:Origin Recognition Complex; D018741:Replication Origin; D012093:Replicon
chemical_list: D004268:DNA-Binding Proteins; D034921:Lamin Type B; D046911:Macromolecular Substances; D051738:Origin Recognition Complex