Different roles of the human Orc6 protein in the replication initiation process
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In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1–Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.
KeywordsOrigin recognition complex Orc6 Chromatin HMGA1a Replication initiation
Bimolecular fluorescent complementation
Dyad symmetry element
Family of repeats
High mobility group
Origin recognition complex
We thank Christoph-Erik Mayer and Stefanie Fülöp for discussions and suggestions and J.F.X. Diffley for the protocol of the plasmid-binding system. We gratefully acknowledge R. Knippers for ideas and advice. This work was supported by the following institutional grants: Deutsche Forschungsgemeinschaft grants SFB646 (A.S.), SFB/TR05 (A.S.), SPP1230 (A.S. and W.H.), and NIH grant CA70723 (W.H.).
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