DOMAIN ANALYSIS OF M ACEIVORANS REPLICATION PROTEIN A 1

M ACEIVORANS 复制蛋白 A 1 的结构域分析

• 批准号: 7357984
• 负责人: ISAAC CANN
• 金额: $ 0.24万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-29 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357984
• 关键词: Archaea; DNA; DNA binding protein; protein structure; proteins

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The oligonucleotide/oligosaccharide-binding (OB) fold, a module ranging from 70-150 amino acids, is central to the architecture of single-stranded DNA-binding proteins. Single-stranded DNA-binding proteins are essential in diverse cellular processes. The bacterial single-stranded DNA-binding protein, a single polypeptide known as SSB, harbors a single OB fold. In contrast, the eukaryotic functional homolog, known as replication protein A (RPA), is a heterotrimeric protein containing multiple OB folds. In the methanogenic archaea, single polypeptide RPA proteins with multiple OB folds and a zinc finger domain have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than the bacterial ones. Here, we describe the functional analysis of a new form of RPA from the methanogenic archaeon Methanosarcina acetivorans. The novel RPA, designated MacRPA1, is comprised of four OB folds and lacks the zinc finger domain hitherto found in the RPA proteins of methanogens. MacRPA1 was compared with two other RPA proteins in M. acetivorans for their effects on DNA strand exchange, DNA synthesis, and cleavage of a flap by cognate proteins involved in these processes. MacRPA1 stimulated DNA strand exchange and primer extension reactions, however, it suppressed cleavage of a flap by the methanosarcinal homolog of flap endonuclease 1. N-terminal and C-terminal truncated derivatives of MacRPA1 were made and their properties were studied using biochemical and biophysical methods. Most interestingly, comparison of MacRPA1 with potential orthologs provided critical insight into a process that may serve to generate new OB folds in cells.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。寡核苷酸/寡糖结合（OB）折叠，一个范围从70-150个氨基酸的模块，是单链DNA结合蛋白质结构的核心。单链DNA结合蛋白在不同的细胞过程中是必不可少的。细菌单链DNA结合蛋白，一种称为SSB的单一多肽，具有单一的OB折叠。相反，真核生物的功能同源物，称为复制蛋白A（RPA），是一种含有多个OB折叠的异源三聚体蛋白。在产甲烷古菌中，已经描述了具有多个OB折叠和锌指结构域的单一多肽RPA蛋白。这些蛋白质的OB折叠比细菌的更类似于它们的真核生物对应物。在这里，我们描述了一种新形式的RPA的产甲烷古菌Methanosarcina acetivorans的功能分析。命名为MacRPA 1的新型RPA由四个OB折叠组成，并且缺乏迄今为止在产甲烷菌的RPA蛋白中发现的锌指结构域。将MacRPA 1与M. acetivorans，因为它们对DNA链交换、DNA合成和通过参与这些过程的同源蛋白切割瓣的作用。MacRPA 1刺激DNA链交换和引物延伸反应，然而，它抑制瓣内切核酸酶1的methanosarcinal同源物的瓣的切割。制备了MacRPA 1的N-末端和C-末端截短衍生物，并使用生物化学和生物物理方法研究了它们的性质。最有趣的是，MacRPA 1与潜在的直系同源物的比较为可能在细胞中产生新的OB折叠的过程提供了关键的见解。