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

近年来，在阐明真核生物DNA复制的分子机制方面取得了相当大的进展。然而，关于人类起源的构成和定义的基本问题仍然是开放的。起源识别的关键参与者是起源识别复合物（origin recognition complex，ORC）。这种蛋白质复合物不仅是DNA复制所必需的，而且对真核细胞染色体周期，即染色质的组织也很重要。目前尚不清楚ORC如何区分这些功能。最近，我们观察到高迁移率族蛋白A（HMGA）不仅与异染色质蛋白1（HP 1）相互作用，而且与ORC共定位和缔合。根据这些调查结果，我们的建议解决了两个主要问题。首先，我们建议用遗传学、生物化学和影像学方法研究HMGA 1a在起源定义和ORC靶向中的功能。特别是，我们的目标是确定由HMGA 1a和ORC识别的DNA元件，并确定这些片段在其天然染色体位置以及附加型系统中的复制能力。将使用突变蛋白和siRNA实验进一步探索功能相关性。此外，我们希望通过使用生物化学和成像方法来指定Orc亚基，HMGA 1和HP 1之间相互作用的分子细节。我们将分析HP 1，HMGA和ORC复合物的动力学，并验证使用共表达和“敲低”策略后，这些复合物的合作和功能。我们的目的是定义的相关性，这种合作的DNA复制和/或异染色质的组织。

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.