Role of HMGA1a and ORC interaction in origin definition and heterochromatin organization

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In recent years considerable progress has been made towards elucidating the molecular mechanisms that control eukaryotic DNA replication. However, the basic question about constitution and definition of human origins is still open. Key player for origin recognition is the origin recognition complex (ORC). This protein complex is not only essential for DNA replication, but also important for the eukaryotic chromosome cycle, i.e. the organization of chromatin. Currently it is not known how ORC discriminates between these functions. Recently, we observed that the high mobility group proteins A (HMGA) not only interact with heterochromatin protein 1 (HP1), but also co-localizes and associates with ORC. Based on these findings our proposal addresses two major questions. Firstly, we propose to study the function of HMGA1a in origin definition and ORC-targeting with genetic, biochemical and imaging approaches. In particular, we aim to identify DNA-elements recognized by HMGA1a and ORC and determine the replication competence of these fragments in their native chromosomal location as well as in episomal systems. The functional relevance will be further explored using mutated proteins and siRNA experiments. Moreover, we wish to specify the molecular details of the interactions between Orc-subunits, HMGA1 and HP1 by using biochemical and imaging approaches. We will analyze the dynamics of HP1, HMGA and ORC complexes and verify the co-operation and function of such complexes after using co-expression and “knock down” strategies. The aim is to define the relevance of such co-operations for DNA replication and/or organization of heterochromatin.