Half a century after their discovery: Structural insights into exonuclease and annealase proteins catalyzing recombineering

Abstract

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.

Keywords

Recombineering / Redβ / λExo / RecE / RecT / Protein structure / Annealase / Exonuclease

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

LJF, CEB, and GT wrote the manuscript. TPN and NPJ performed the sequence analysis on RecE and λExo exonuclease families, prepared Fig.4, and wrote the text for Section 2.4 and Section 4.1. CEB prepared Fig. 2 and Fig. 3. LJF prepared all other figures and tables.

Acknowledgments

This work was funded by the National Science Foundation Grant MCB-2212951 (to CEB) and NHMRC Ideas grant APP1184012/GNT1184012 (to GT).

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