Structure/function of RecA protein from P. aeruginosa

描述(申请人提供)这项研究将主要在俄罗斯圣彼得堡的彼得堡核物理研究所与弗拉迪斯拉夫·兰佐夫合作完成，作为NIH资助号R01GM32335的延伸。大肠杆菌RecA蛋白在体外促进了DNA链交换反应，为重组DNA修复和同源基因重组的中心步骤提供了一个方便的分子模型。R01 GM32335(COX)研究的长期目标是详细了解RecA介导的DNA链交换。重组DNA修复是RecA蛋白在体内的主要功能的假设提供了一个智力框架。GM32335-21(最近资助)的具体目标之一是了解RecA功能是如何自动调节的。铜绿假单胞菌是一种重要的人类病原体，它是一种RecA蛋白的来源，为了进一步实现这一目标，我们将对其进行研究。我们建议继续进行富有成效的合作，通过对铜绿假单胞菌RecA蛋白的分析，扩大我们对RecA自动调节的理解。迄今为止的工作提供了许多机械性和结构性的见解，我们将在此基础上再接再厉。我们将与俄罗斯圣彼得堡的Vladislava Lanzov博士的实验室一起，进一步探索铜绿假单胞菌RecA蛋白的生物化学，并确定它与大肠杆菌RecA之间的酶学差异。我们已经确定了负责自动调节的蛋白质的可能区域，以及RecA蛋白质之间的某些其他功能差异。这项新工作应该有助于测试RecA介导的DNA链交换模型的关键功能，也可能有助于识别具有增强的DNA结合和链交换功能的RecA变体。这些蛋白质最终可能被证明是有用的，在基因治疗方案中使用RecA，并产生用于结构分析的RecA-DNA复合体的晶体。

DESCRIPTION (provided by applicant) This research will be done primarily in St. Petersburg, Russia at Petersburg Nuclear Physics Institute in collaboration with Vladislav Lanzov as an extension of NIH grant Number R01GM32335. The RecA protein of E. coli promotes a DNA strand exchange reaction in vitro that provides a convenient molecular model for the central steps of recombinational DNA repair and homologous genetic recombination. The long-range goal of the research in R01 GM32335 (Cox) is a detailed understanding of RecA-mediated DNA strand exchange. The hypothesis that recombinational DNA repair is the primary function of RecA protein in vivo provides an intellectual framework. One of the specific aims of GM32335-21 (recently funded) is to understand how RecA function is autoregulated. Pseudomonas aeruginosa, an important human pathogen, is the source of a RecA protein that we will investigate to further this aim. We propose to continue a productive collaboration that has been extending our understanding of RecA autoregulation through an analysis of the Pseudomonas aeruginosa RecA protein. Work to date has provided numerous mechanistic and structural insights that we will build on. Together with the laboratory of Dr. Vladislava Lanzov in St. Petersburg, Russia, we will further explore the biochemistry of the P. aeruginosa RecA protein, and define the enzymatic differences between it and the E. coli RecA. We have pinpointed likely regions of the protein responsible for autoregulation and for certain other functional differences between the RecA proteins. The new work should help test key features of models for RecA-mediated DNA strand exchange, and may also help identify RecA variants with enhanced DNA binding and strand exchange functions. Such proteins may eventually prove useful in efforts to use RecA in gene therapy protocols and to generate crystals of RecA-DNA complexes for structural analysis.