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  • Half a century after their discovery: Structural insights into exonuclease and annealase proteins catalyzing recombineering
Title: Half a century after their discovery: Structural insights into exonuclease and annealase proteins catalyzing recombineering
Recombineering is an essential tool for molecular biologists, allowing for the facile and efficient manipulation of bacterial genomes directly in cells without the need for costly and laborious in vitro manipulations involving restriction enzymes. The main workhorses behind recombineering are bacteriophage proteins that promote the single-strand annealing (SSA) homologous recombination pathway to repair double-stranded DNA breaks. While there have been several reviews examining recombineering methods and applications, comparatively few have focused on the mechanisms of the proteins that are the key players in the SSA pathway: a 5′→3′ exonuclease and a single-strand annealing protein (SSAP or “annealase”). This review dives into the structures and functions of the two SSA recombination systems that were the first to be developed for recombineering in E. coli: the RecET system from E. coli Rac prophage and the λRed system from bacteriophage λ. By comparing the structures of the RecT and Redβ annealases, and the RecE and λExo exonucleases, we provide new insights into how the structures of these proteins dictate their function. Examining the sequence conservation of the λExo and RecE exonucleases gives more profound insights into their critical functional features. Ultimately, as recombineering accelerates and evolves in the laboratory, a better understanding of the mechanisms of the proteins behind this powerful technique will drive the development of improved and expanded capabilities in the future.  more » « less
Award ID(s):
2212951
PAR ID:
10517137
Author(s) / Creator(s):
; ; ; ;
Publisher / Repository:
Elsevier
Date Published:
Journal Name:
Engineering Microbiology
Volume:
4
Issue:
1
ISSN:
2667-3703
Page Range / eLocation ID:
100120
Subject(s) / Keyword(s):
Recombineering Redβ λExo RecE RecT Protein structure Annealase Exonuclease
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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