Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA.
Adv Virus Res. 2012;83:367-414. doi: 10.1016/B978-0-12-394438-2.00008-6.
The homologous recombination systems of linear double-stranded (ds)DNA bacteriophages are required for the generation of genetic diversity, the repair of dsDNA breaks, and the formation of concatemeric chromosomes, the immediate precursor to packaging. These systems have been studied for decades as a means to understand the basic principles of homologous recombination. From the beginning, it was recognized that these recombinases are linked intimately to the mechanisms of phage DNA replication. In the last decade, however, investigators have exploited these recombination systems as tools for genetic engineering of bacterial chromosomes, bacterial artificial chromosomes, and plasmids. This recombinational engineering technology has been termed "recombineering" and offers a new paradigm for the genetic manipulation of bacterial chromosomes, which is far more efficient than the classical use of nonreplicating integration vectors for gene replacement. The phage λ Red recombination system, in particular, has been used to construct gene replacements, deletions, insertions, inversions, duplications, and single base pair changes in the Escherichia coli chromosome. This chapter discusses the components of the recombination systems of λ, rac prophage, and phage P22 and properties of single-stranded DNA annealing proteins from these and other phage that have been instrumental for the development of this technology. The types of genetic manipulations that can be made are described, along with proposed mechanisms for both double-stranded DNA- and oligonucleotide-mediated recombineering events. Finally, the impact of this technology to such diverse fields as bacterial pathogenesis, metabolic engineering, and mouse genomics is discussed.
线性双链 (ds)DNA 噬菌体的同源重组系统对于产生遗传多样性、修复 dsDNA 断裂以及形成连环染色体（包装的直接前体）是必需的。这些系统作为理解同源重组基本原理的一种手段已经研究了几十年。从一开始，就认识到这些重组酶与噬菌体 DNA 复制的机制密切相关。然而，在过去的十年中，研究人员已经将这些重组系统作为细菌染色体、细菌人工染色体和质粒的遗传工程工具加以利用。这种重组工程技术被称为“重组酶工程”，为细菌染色体的遗传操作提供了一个新的范例，其效率远远高于经典的非复制整合载体用于基因替换。特别是噬菌体 λ Red 重组系统已被用于构建大肠杆菌染色体中的基因替换、缺失、插入、倒位、重复和单碱基对变化。本章讨论了 λ、rac 噬菌体和噬菌体 P22 的重组系统的组成部分，以及来自这些噬菌体和其他噬菌体的单链 DNA 退火蛋白的特性，这些特性对于该技术的发展至关重要。描述了可以进行的遗传操作类型，并提出了双链 DNA 和寡核苷酸介导的重组酶工程事件的机制。最后，讨论了这项技术对细菌发病机制、代谢工程和小鼠基因组学等不同领域的影响。
