重组工程
生物
重组酶
遗传学
DNA
核酸外切酶
重组
核酸外切酶 III
细菌圆形染色体
大肠杆菌
DNA复制
质粒
FLP-FRT重组
λ噬菌体
校对
分子生物学
基因
遗传重组
噬菌体
DNA聚合酶
作者
Joshua A. Mosberg,Marc J. Lajoie,George M. Church
出处
期刊:Genetics
[Oxford University Press]
日期:2010-09-03
卷期号:186 (3): 791-799
被引量:187
标识
DOI:10.1534/genetics.110.120782
摘要
Abstract The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5′ ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3′ overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.
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