Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins

Abstract

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G 1 and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.chromatin ͉ DNA replication ͉ EBNA1 ͉ Epstein-Barr virus ͉ Orc6

Functional analysis of an Orc6 mutant in Drosophila

“…We have observed similar staining patterns for other Drosophila ORC subunits, including Orc1 and Orc2. Moreover, ORC was found in the nucleolus in human cells (31,32). These findings may indicate a potential interaction of ORC with components of the ribosome biogenesis pathway (31,33).…”

Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB

“…Human Orc6 associates with chromatin in human cell lines (27,28). Moreover, human Orc6 partially restores DNA replication when expressed in Drosophila mutant cells carrying the deletion of orc6 gene (20).…”

“…Several examples include E2F and Myb transcription factors, which interact with ORC and target it to the origin at chorion amplification locus in Drosophila (35,36). Recent studies revealed the role of HMG1A and WD repeat-containing LRWD1 proteins in targeting of ORC to the chromatin in human cells (28,37). Interestingly, human Orc6 and HMG1A proteins interact directly and may both contribute to the targeting of ORC to the sites of replication initiation and to the pre-RC assembly.…”

“…In human cells, Orc6 localizes in the nucleus together with other ORC subunits and also associates with chromatin (27,28). Based on our structural studies, we asked if human Orc6, similar to its Drosophila homolog, could interact with DNA directly.…”

Comment l’approche génomique aide à comprendre le processus d’initiation de la réplication

“…[20][21][22] qui est essentielle pour l'assemblage du complexe pré-RC au cours de la phase G1 et l'initiation de la réplication en phase S [20,22]. Il est donc envisageable que CDT1, en coopération avec certains facteurs de transcription, puisse définir un site d'ancrage, et de régulation, de HBO1 au niveau des origines de réplication [17][18][19][20][21][22]. Il a aussi été démontré que les voies de signalisation qui régulent la transcription agissent également sur les constituants du complexe pré-RC pour contrôler la réplication [23].…”