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.In 1963 Jacob, Brenner, and Cuzin (24) proposed the replicon model to explain the control of replication of the bacterial chromosome. In this model, DNA replication starts from a specific origin sequence, the replicator, that is recognized by a positive regulatory protein, the initiator. Since then the model has been validated in numerous prokaryotic and viral systems. The organization of the eukaryotic genome in multiple replication units distributed on several chromosomes has hampered the validation of this model in eukaryotes until the identification of the autonomous replicating sequences (ARS) in Saccharomyces cerevisiae. Initially identified for their ability to support the propagation of plasmid molecules in yeast cells, most of these sequences were successively proven to correspond to chromosomal replicators. ARSs are relatively short sequence elements (100 to 200 bp) that include the start site of replication, also called the origin of bidirectional DNA replication (OBR) (11). They consist of an essential 11-bp ARS consensus sequence (ACS) and of several auxiliary B elements that contribute to initiation activity. The ACS binds the origin recognition complex (ORC), a heteromeric complex of six proteins that assists the formation of a prereplicative complex on the origin. ORC orthologs have been isolated from all the eukaryotic species analyzed so far, including humans (for a review see reference 8). The ARS sequences and the ORC can be viewed as the prototypes of the eukaryotic replicator and initiator.The major obstacle to the validation of the replicon model in metazoan cells was the failure to isolate the functional homologues of the ARS elements...

Genome-wide studies highlight indirect links between human replication origins and gene regulation

“…Because not all active promoters are efficient sites of replication initiation, active promoters containing an origin must have distinguishing information. The fact that cis-elements containing known origins remain strong sites of replication initiation even when inserted ectopically also suggests that specific replication start points are recognized by the replication machinery (32,33). We speculate that the replication machinery efficiently recognizes specific combinations of transcription factors.…”

Discrete functional elements required for initiation activity of the Chinese hamster dihydrofolate reductase origin beta at ectopic chromosomal sites

“…The observation that elements >3 kb from the ori-beta initiation site are critical for ori-beta activity is perhaps surprising given that a 1.2 kb fragment from the human LaminB2 origin is sufficient for ectopic initiation activity [21]. However, such a distal effect is not unprecedented given that deletion of the distal hamster DHFR promoter affects replication initiation in the entire downstream 55 kb intergenic region [46].…”

“…To achieve this, two general strategies have been used. Using a non-specific integration system, the 5.8 kb Chinese hamster DHFR origin beta (ori-beta) [8,9] and the 1.2 kb human LaminB2 origin [21] showed replication initiation activity at multiple chromosomal sites in mammalian cells. These functioned as active replicators in pooled stably transfected cells or individual cell clones, although DNA replication activity varied at different chromosomal sites.…”

Replication Timing: Evolution, Nuclear Organization and Relevance for Human Disease

“…Fission yeast and Drosophila have ORCs that recognize AT-rich sequences (Austin et al, 1999;Chuang and Kelly, 1999), rather than specific motifs. Moreover, human ORCs, which are chosen as initiators of replication, have also been shown to require AT-rich sequences as well as various other features, including matrix attachment region sequences, dinucleotide repeats and asymmetrical purine-pyrimidine sequences (Altman and Fanning, 2004;Debatisse et al, 2004;Paixao et al, 2004;Schaarschmidt et al, 2004;Wang et al, 2004). Other factors that may affect the initiation of replication at certain ORs also include DNA topology, transcription factors, and elements of the pre-replicative complex (pre-RC) (reviewed in Masai et al, 2010).…”