Assembly of Replicative Complexes

说明书(申请人提供)：大肠杆菌的DNA聚合酶III全酶是一个典型的复制复合体，表现出与其他细胞复制酶的共同性质，包括高速率的过程延伸和在复制分叉处与其他蛋白质相互作用的能力，建立协调有效染色体复制所需事件所需的通信通道。所有细胞复制酶的一个关键组成部分是同源蛋白的多亚基组装，需要ATP将“滑动钳制加工性因子”组装到引物末端。在大肠杆菌中，这一功能是由DNAX复合体DnaX3deltadelta‘/khi/psi提供的。大肠杆菌的DNA X基因编码两种不同的产物：I，全长翻译产物和伽马，一种通过翻译移码而产生的较短的蛋白质。在下一个资助期间，我们将研究i)DNA聚合酶III全酶的组装途径，重点是POL III在引导组装途径以允许DNAX亚基的独特排列中的作用，ii)确定通过DNAX复合体‘钳夹加载器’将Beta2滑动夹加载到已启动的DNA过程中发生的大分子相互作用的变化，iii)确定ATPGammaS辅助的起始复合体形成的机制，以及iv)研究DNAX在起始复合体形成中的类似伴侣的特性。

DESCRIPTION (provided by applicant) The DNA polymerase III holoenzyme of E. coli is a prototypical replicative complex, exhibiting properties in common with other cellular replicases, including a high rate of processive elongation and the ability to interact with other proteins at the replication fork, establishing the communication channels necessary to coordinate the events required for efficient chromosomal replication. A key component of all cellular replicases is a multisubunit assembly of homologous proteins that require ATP to assemble a "sliding clamp processivity factor" onto primer termini. In E. coli, this function is served by the DnaX complex, DnaX3deltadelta'/khi/psi. The dna X gene of E. coli encodes two distinct products: i, the full-length translation product and gamma, a shorter protein that arises by translational frameshifting. During the next grant period, we will study i) the assembly pathway of DNA polymerase III holoenzyme, with emphasis on the role of Pol III in steering the assembly pathway to permit a unique arrangement of DnaX subunits, ii) determine the changes in macromolecular interactions that occur during loading of the beta2 sliding clamp onto primed DNA by the DnaX complex 'clamp loader,' iii) determine the mechanism of ATPgammaS-assisted initiation complex formation that proceeds without nucleotide hydrolysis and iv) study the chaperone-like properties of DnaX in initiation complex formation.