Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants

Abstract

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage -encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20 -70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence. H omologous recombination mediated by Red has been used as a genetic tool to modify the bacterial chromosome with linear double-stranded DNA (dsDNA) (1-6). In addition to linear dsDNA, single-stranded DNA (ssDNA) oligonucleotides (oligos) have been used to modify the chromosomes of both yeast and Escherichia coli (7-9). In yeast, the functions are not yet defined that allow oligo recombination; however, in E. coli, the bacteriophage Red recombination system is involved (9).The Red system includes the Gam, Exo, and Beta proteins. Whereas Red-mediated recombination between linear duplex DNA and the bacterial chromosome requires all three functions (1, 3, 10), recombination with ssDNA oligos requires only the Beta protein (9). Beta protein binds stably to ssDNA (11) Ͼ35 nt in length (12), protects it from single-strand nuclease attack (13,14), and promotes annealing to complementary ssDNA (13-15).Beta binds ssDNA from 3Ј to 5Ј (13, 16) but does not bind directly to dsDNA (13,14). However, after Beta generates dsDNA by annealing two complementary ssDNAs, it remains tightly bound to the annealed dsDNA (13,14,16). As it anneals strands to form the dsDNA, it generates DNA filaments similar to those formed by . This annealed dsDNA-Beta complex is resistant to DNase I and is much more stable than the ssDNA-Beta complex (16).Although Beta can catalyze single-strand annealing, it cannot promote strand invasion of a duplex DNA with a homologous ssDNA during recombination (16,18). Therefore, Betamediated recombination with ssDNA is likely to occur by annealing with transiently single-stranded regions of the chromosome. Initial results suggest that Beta-dependent ssDNA recombination may occur at the DNA replication fork. When either of the two complementary ssDNA oligos...

Multicopy plasmid modification with phage λ Red recombineering

“…These experiments confirm the early obervations of others (Zhang et al, 1998;Yu et al, 2000): recombineering can be as useful a tool for modifying plasmid replicons as it is for BACs Muyrers et al, 1999), the bacterial chromosome (Yu et al, 2000;Ellis et al, 2001;Costantino and Court, 2003;Thomason et al, 2005b), and phage λ itself (Oppenheim et al 2004), but there are complexities associated with multicopy plasmids that do not arise with the other replicons. ssDNA recombineering in the absence of mismatch repair targeted to plasmids is of such high efficiency that it can be used to isolate point mutations or changes of only a few bases without selection.…”

“…This reaction was performed in recA mutant strains either expressing all three Red genes exo, bet, and gam or expressing only the Red bet gene. Deletion of a large DNA segment is not affected by mismatch repair (Costantino and Court, 2003;Parker and Marinus, 1992; and our unpublished data) and the lagging-leading differential is apparent in this reaction, with the lagging-strand oligo frequency 10-to 30-fold higher than that of the leading-strand oligo (Table 5). Both reactions were substantially lower in efficiency than the corresponding frequency observed for repair of a point mutation with these oligos in the absence of mismatch repair (compare Table 3 and Table 5).…”

“…To examine recombineering in the absence of mismatch repair, either mismatch repair was eliminated by mutation of mutS; or the oligo was designed so that, when bound to the target, it created a C-C mismatch that is not subject to mismatch repair even in the presence of an active MMR system (Costantino and Court, 2003). Both methods gave similar results.…”

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…While the first study focused only on RecT of Rac prophage, in the second, authors detected high rates of recombineering activity in E. coli from an array of recombinases originating from both gram‐negative and gram‐positive bacteria (10 −4 to 10 −1 mutants/viable cell). While such rates of allelic exchange reflect an increase in two orders of magnitude from the numbers we have observed (10 −6 to 10 −3 mutants/viable cell), we note the use of an E. coli ∆ mutS strain devoid of mismatch repair machinery, which could have increased recombineering rates by >100‐fold (Costantino and Court, 2003). In contrast, P. putida EM42 – a streamlined derivative of P. putida KT2440 (Martínez‐García et al ., 2014) – contains a fully intact MMR system, making a direct, quantitative study comparison difficult.…”

“…1B. In keeping with established strand bias and previous study design (Ellis et al ., 2001; Costantino and Court, 2003; Aparicio et al ., 2016), SR was targeted to the lagging strand of the P. putida rpsL gene and engineered to evade endogenous MMR activity, in order to maximize putative recombinase efficiency. Further information on the design and synthesis of the mutagenic oligonucleotide can be found in the Experimental procedures section.…”