Mechanisms and Application Research Progress of Bacterial Genomic Homologous Recombination Mediated by Single-stranded DNA Annealing Protein

doi:10.13560/j.cnki.biotech.bull.1985.2024-0457

Abstract

Abstract:

Genome editing technology is an important tool for studying the gene function, drug resistance mechanism, and pathogenic mechanism of bacteria and other microorganisms. Homologous recombination is one of the key methods for bacterial genome editing. The conventional endogenous pathways in bacteria suffer from low efficiency. However, a single-stranded DNA annealing protein（SSAP）from bacteriophages, which has shown gene editing efficiencies far surpassing those of endogenous pathways. This protein has the characteristics of single-stranded DNA binding activity and mediates genome directed recombination, making it an extremely promising tool for genome editing. This article provides an overview of the basic principles of homologous recombination, the fundamental components of the SSAP-mediated homologous recombination pathway from phages, recombination mechanism models, and application strategies. The aim is to assist in further elucidating the process of homologous recombination mediated by SSAP to provide technical support for studying the functions and pathogenic mechanisms of more bacterial genes, and to develop engineering strains. It also provides technical support for bacteria lacking gene editing methods.

Key words: bacteria, single-stranded DNA annealing protein, homologous recombination, mechanism, application

YIN Hao, YOU Liu-chao, HAN Rui, GAO Peng-cheng, FU Lei, CHU Yue-feng. Mechanisms and Application Research Progress of Bacterial Genomic Homologous Recombination Mediated by Single-stranded DNA Annealing Protein[J]. Biotechnology Bulletin, 2025, 41(1): 39-48.

Figures/Tables 2

Fig. 1 Model of RecA-mediated recombination of ssDNA and the strand annealing model of SSAP RecA possesses ssDNA-binding activity, SSAP has ssDNA annealing activity, and Exo has 5'-3' exonuclease activity targeting dsDNA. The homologous recombination process mediated by RecA is primarily involved in strand invasion, while the homologous recombination process mediated by SSAP primarily in strand annealing. a: RecA binds to ssDNA to form a RecA-DNA complex, which carries the ssDNA to invade the homologous sequence in the genome, forming a D-loop structure, thereby completing the recombination process. b: SSAP can directly bind to ssDNA, annealing it to the replication fork in the form of Okazaki fragments. dsDNA is processed into ssDNA by Exo and then completes recombination in the same manner

Fig. 2 Schematic diagram of the recombination process mediated by SSAP SSAP possesses ssDNA annealing activity, while Exo exhibits a 5'-3' exonuclease activity targeted at dsDNA. a: SSAP mediates the annealing of ssDNA, annealing it to the lagging strand in the form of Okazaki fragments. b: dsDNA is processed by Exo into either a completely degraded single strand of ssDNA or a recombination intermediate exposing 3' overhangs at both ends. c: The generally accepted model of SSAP-mediated ssDNA recombination strategy at the replication fork. d: A cooperative working model where Exo degrades one strand of the dsDNA while the other strand is simultaneously annealed by SSAP into the replication fork

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