DOMAIN ANALYSIS OF M ACEIVORANS REPLICATION PROTEIN A 1

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。寡核苷酸/寡糖结合（OB）折叠，一个包含70-150个氨基酸的模块，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是必不可少的。细菌单链dna结合蛋白是一种被称为SSB的多肽，含有一个OB折叠。相反，真核生物的功能同源物，被称为复制蛋白A (RPA)，是一种含有多个OB折叠的异源三聚体蛋白。在产甲烷的古菌中，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。这些蛋白的OB折叠与真核生物的OB折叠比细菌的OB折叠更相似。本文对产甲烷古菌活性甲烷古菌（Methanosarcina etivorans）中一种新的RPA进行了功能分析。新的RPA被命名为MacRPA1，由四个OB折叠组成，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MacRPA1与m.a actitivorans中的其他两种RPA蛋白进行比较，以了解它们对DNA链交换、DNA合成以及参与这些过程的同源蛋白对皮瓣切割的影响。MacRPA1刺激了DNA链交换和引物延伸反应，但它抑制了皮瓣内切酶1的甲烷同源物对皮瓣的切割。制备了MacRPA1的n端和c端截断衍生物，并采用生物化学和生物物理方法对其性质进行了研究。最有趣的是，将MacRPA1与潜在的同源基因进行比较，对细胞中可能产生新的OB折叠的过程提供了关键的见解。

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.