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

近年来，在阐明控制真核DNA复制的分子机制方面取得了相当大的进展。然而，关于宪法和人类起源定义的基本问题仍然悬而未决。起源识别的关键角色是起源识别复合体(ORC)。这种蛋白质复合体不仅对DNA复制是必不可少的，而且对真核细胞的染色体周期也是重要的，即染色质的组织。目前还不知道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(HP1)相互作用，而且与ORC共定位和结合。基于这些发现，我们的提案解决了两个主要问题。首先，我们建议从遗传学、生物化学和成像的角度研究HMGA1a在起源定义和ORC靶向中的作用。特别是，我们的目标是鉴定HMGA1a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在上体系统中的复制能力。将通过突变蛋白质和siRNA实验进一步探索功能相关性。此外，我们希望通过生化和成像方法详细说明ORC-亚基、HMGA1和HP1之间的相互作用的分子细节。我们将分析HP1、HMGA和ORC复合体的动力学，并验证这些复合体在使用共表达和“击倒”策略后的合作和功能。目的是确定这种合作与DNA复制和/或异染色质组织的相关性。

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.