Mechanism of Homologous Recombination in Archaea

同源重组是化学诱变剂和辐射引起的双链断裂修复的主要机制。修复这些断裂是进化的必要条件，可以确保信息在不丢失的情况下从一代传递到另一代。对这一过程的一般理解主要来自对细菌或真核生物分支的研究。我们对第三个古细菌分支的双链断裂修复知之甚少。古细菌在物理上与细菌相似，但使用一些细胞方式来生活，似乎更像真核生物。这些真核类过程的关键是基因组维护方法，包括双链断裂修复。这提供了一个独特的机会来研究同源重组机制在一个进化上不同的生物群体。该项目的总体目标是利用已建立的古细菌模型Sulfolobus solfataricus来确定三种与链交换蛋白RadA序列相关的蛋白质的机制作用。这些类似物的研究将涉及研究生，本科生和高中生，使用两种互补但独立的方法：1)蛋白质在体外的生化活性将被单独确定，并与其他已知的重组相关蛋白质结合，其中它们的相互作用和修复途径中的功能点将被建立；2)在野生型和突变型菌株背景下，通过研究辐射损伤后蛋白质的持久性和测量染色体修复率来评估其在体内的作用。这个项目的重点是在DNA损伤后细胞存活的必要机制，这是研究最少的生命分支的成员。除了更多地了解所有细胞修复DNA的方式外，这项工作有望为同源重组的调控和基因组稳定性的维持提供新的整体见解。这些研究是多学科的，使用生化、遗传和细胞方法，将为不同教育阶段的学生提供广泛的实验室教育。具体而言，该项目为一名研究生和两名本科生提供实验室培训，并特别强调对代表性不足的少数民族学生的外展招募。此外，两名高中生将通过暑期实习得到支持，这将为处于职业生涯早期阶段的年轻科学家提供宝贵的研究经验。

Homologous recombination is a major mechanism for repair of double-strand breaks caused by exposure to chemical mutagens and radiation. Repair of these breaks is an evolutionary imperative and ensures proper transfer of information from generation to generation without information loss. General understanding of this process has come mostly from studies from the bacterial or eukaryotic branches of life. Very little is known about double-strand break repair in the third, archaeal branch. Archaea physically resemble bacteria, but use some cellular approaches to life that seem to be more eukaryotic. Key among these eukaryotic-like processes are methods of genome maintenance, including double-strand break repair. This provides a unique opportunity to study homologous recombination mechanisms in an evolutionarily distinct group of organisms. The overall objective of this project is to use the well established archaeal model Sulfolobus solfataricus to determine the mechanistic role of three proteins that are related by sequence to the strand-exchange protein, RadA. The research on these paralogues will involve graduate, undergraduate, and high school students using two complementary but independent approaches: 1) Biochemical activity of the proteins in vitro will be determined individually and in combination with other known recombination-related proteins, where their interactions and point of function in the repair pathway will be established; 2) Roles of the paralogues in vivo will be assessed through studies of protein persistence after radiation damage and measurement of chromosomal repair rates in both wild-type and mutant strain backgrounds.This project focuses on a mechanism necessary for cellular survival following DNA damage in a member of the least well studied branch of life. In addition to understanding more about the way in which all cells repair their DNA, this work is expected to provide new overall insight into the regulation of homologous recombination and maintenance of genome stability. These studies are multidisciplinary, using biochemical, genetic, and cellular approaches that will provide a broad laboratory education for students at various educational stages. Specifically, this project provides laboratory training for one graduate and two undergraduate students, with a special emphasis on outreach recruiting of underrepresented minority students. Additionally, two high school students will be supported through summer internships, which will offer a valuable research experience to young scientists at early stages in their careers.