DOMAIN ANALYSIS OF M ACEIVORANS REPLICATION PROTEIN A 1

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。寡核苷酸/寡糖结合(OB)折叠是单链DNA结合蛋白结构的核心，它是一个由70-150个氨基酸组成的模块。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结合蛋白是一种被称为SSB的单一多肽，它含有一个OB折叠。相比之下，真核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。在产甲烷古生菌中，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。这些蛋白的OB折叠比细菌的OB折叠更接近于它们的真核对应蛋白。在这里，我们描述了一种新形式的RPA的功能分析，这些RPA来自产甲烷的古生菌--乙酸甲烷八叠球菌。新的RPA被命名为MacRPA1，由四个OB折叠组成，缺乏迄今在产甲烷菌的RPA蛋白中发现的锌指结构域。比较了MacRPA1和其他两种在食性支原体中的RPA蛋白对DNA链交换、DNA合成和参与这些过程的同源蛋白对瓣膜切割的影响。MacRPA1可刺激DNA链交换和引物链延伸反应，但它能抑制FET-1的甲烷糖同系物对瓣膜的切割作用。最有趣的是，将MacRPA1与潜在的同源基因进行比较，提供了对可能在细胞中产生新的OB折叠的过程的关键洞察。

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.