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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。寡核苷酸/寡糖结合 (OB) 折叠是一个由 70-150 个氨基酸组成的模块，是单链 DNA 结合蛋白结构的核心。单链 DNA 结合蛋白在多种细胞过程中至关重要。细菌单链 DNA 结合蛋白是一种称为 SSB 的单一多肽，具有单个 OB 折叠。相比之下，真核功能同源物，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多肽 RPA 蛋白。这些蛋白质的 OB 折叠与真核生物的对应物比与细菌的相似。在这里，我们描述了来自产甲烷古菌 Methanosarcina acetivorans 的一种新型 RPA 的功能分析。这种新型 RPA 被命名为 MacRPA1，由四个 OB 折叠组成，并且缺乏迄今为止在产甲烷菌 RPA 蛋白中发现的锌指结构域。将 MacRPA1 与 M. acetivorans 中的其他两种 RPA 蛋白进行比较，了解它们对 DNA 链交换、DNA 合成以及参与这些过程的同源蛋白裂解瓣的影响。 MacRPA1 刺激 DNA 链交换和引物延伸反应，然而，它抑制瓣膜内切核酸酶 1 的甲烷八叠球菌同源物对瓣膜的切割。制备了 MacRPA1 的 N 端和 C 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特性。最有趣的是，MacRPA1 与潜在直系同源物的比较提供了对可能有助于在细胞中生成新 OB 折叠的过程的重要见解。