Single-strand Annealing Protein Research Articles

Structural Basis for the Interaction of Redβ Single-Strand Annealing Protein with Escherichia coli Single-Stranded DNA-Binding Protein

Redβ is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redβ is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redβ can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redβ with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redβ and SSB involves the C-terminal domain (CTD) of Redβ and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redβ CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redβ and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redβ-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.

Recombineering using RecET-like recombinases from Xenorhabdus and its application in mining of natural products.

Xenorhabdus can produce a large number of secondary metabolites with insecticidal, bacteriostatic, and antitumor activities. Efficient gene editing tools will undoubtedly facilitate the functional genomics research and bioprospecting in Xenorhabdus. In this study, BlastP analysis using the amino acid sequences of Redαβ or RecET recombinases as queries resulted in the identification of an operon (XBJ1_operon 0213) containing RecET-like recombinases encoding genes from the genome of Xenorhabdus bovienii strain SS-2004. Three proteins encoded by this operon was indispensable for full activity of recombineering, namely XBJ1-1173 (RecE-like protein), XBJ1-1172 (RecT-like protein), and XBJ1-1171 (single-strand annealing protein). Using this newly developed recombineering system, a gene cluster responsible for biosynthesis of a novel secondary metabolite (Min16) was identified from X. stockiae HN_xs01 strain. Min16 which exhibited antibacterial and cytotoxic activities was determined to be a cyclopeptide composed of Acyl-Phe-Thr-Phe-Pro-Pro-Leu-Val by using high-resolution mass spectrometry and nuclear magnetic resonance analysis, and was designated as changshamycin. This host-specific recombineering system was proven to be effective for gene editing in Xenorhabdus, allowing for efficient discovery of novel natural products with attractive bioactivities. KEY POINTS: • Screening and identification of efficient gene editing tools from Xenorhabdus • Optimization of the Xenorhabdus electroporation parameters • Discovery of a novel cyclopeptide compound with multiple biological activities.

DCas9-based gene editing for cleavage-free genomic knock-in of long sequences

Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR–Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9–SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9–SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9–SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.

Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria.

Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional mutagenesis at multiple sites on kilobase scales in diverse bacteria without the generation of double-strand breaks or disturbance of the mismatch repair system. dReaMGE can achieve combinatorial genome engineering works, for example, alterations to multiple biosynthetic pathways, multiple promoter or gene insertions, variations of transcriptional regulator combinations, within a few days. dReaMGE adds to the repertoire of bacterial genome engineering to facilitate discovery, functional genomics, strain optimization and directed evolution of microbial cell factories.

Mutational Analysis of Redβ Single Strand Annealing Protein: Roles of the 14 Lysine Residues in DNA Binding and Recombination In Vivo.

Redβ is a 261 amino acid protein from bacteriophage λ that promotes a single-strand annealing (SSA) reaction for repair of double-stranded DNA (dsDNA) breaks. While there is currently no high-resolution structure available for Redβ, models of its DNA binding domain (residues 1–188) have been proposed based on homology with human Rad52, and a crystal structure of its C-terminal domain (CTD, residues 193-261), which binds to λ exonuclease and E. coli single-stranded DNA binding protein (SSB), has been determined. To evaluate these models, the 14 lysine residues of Redβ were mutated to alanine, and the variants tested for recombination in vivo and DNA binding and annealing in vitro. Most of the lysines within the DNA binding domain, including K36, K61, K111, K132, K148, K154, and K172, were found to be critical for DNA binding in vitro and recombination in vivo. By contrast, none of the lysines within the CTD, including K214, K245, K251, K253, and K258 were required for DNA binding in vitro, but two, K214 and K253, were critical for recombination in vivo, likely due to their involvement in binding to SSB. K61 was identified as a residue that is critical for DNA annealing, but not for initial ssDNA binding, suggesting a role in binding to the second strand of DNA incorporated into the complex. The K148A variant, which has previously been shown to be defective in oligomer formation, had the lowest affinity for ssDNA, and was the only variant that was completely non-cooperative, suggesting that ssDNA binding is coupled to oligomerization.

Expanding Diversity of Firmicutes Single-Strand Annealing Proteins: A Putative Role of Bacteriophage-Host Arms Race.

Bacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases which can substitute the classical, bacterial RecA and manage the DNA metabolism at different steps of phage propagation. SSAPs have been shown to efficiently promote recombination between short and rather divergent DNA sequences and were exploited for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the conserved and almost universal bacterial RecA protein, SSAPs display great sequence diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is underlined by their role as key players in events of horizontal gene transfer (HGT). All of the above provoke a constant interest for the identification and study of new phage recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body of putative ssap genes escapes conventional classification, as they are not properly annotated. In this work, we performed a wide-scale identification, classification and analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using sequence similarity network and gene context analyses, we created a new high quality dataset of phage-related SSAPs, substantially increasing the number of annotated SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA, Gp2.5, RecT/Redβ, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies. Analysis of the relationships between the revealed protein clusters led us to recognize Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular phylogenetic distribution of ssap genes among different bacterial phyla and specific phages, which can be explained by the high rates of ssap HGT. We propose that the evolution of phage recombinases could be tightly linked to the dissemination of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas systems) targeting ssap genes and be a part of the constant phage-bacteria arms race.

Oligomeric complexes formed by Redβ single strand annealing protein in its different DNA bound states.

Redβ is a single strand annealing protein from bacteriophage λ that binds loosely to ssDNA, not at all to pre-formed dsDNA, but tightly to a duplex intermediate of annealing. As viewed by electron microscopy, Redβ forms oligomeric rings on ssDNA substrate, and helical filaments on the annealed duplex intermediate. However, it is not clear if these are the functional forms of the protein in vivo. We have used size-exclusion chromatography coupled with multi-angle light scattering, analytical ultracentrifugation and native mass spectrometry (nMS) to characterize the size of the oligomers formed by Redβ in its different DNA-bound states. The nMS data, which resolve species with the highest resolution, reveal that Redβ forms an oligomer of 12 subunits in the absence of DNA, complexes ranging from 4 to 14 subunits on 38-mer ssDNA, and a much more distinct and stable complex of 11 subunits on 38-mer annealed duplex. We also measure the concentration of Redβ in cells active for recombination and find it to range from 7 to 27 μM. Collectively, these data provide new insights into the dynamic nature of the complex on ssDNA, and the more stable and defined complex on annealed duplex.

Microbial single-strand annealing proteins enable CRISPR gene-editing tools with improved knock-in efficiencies and reduced off-target effects.

Several existing technologies enable short genomic alterations including generating indels and short nucleotide variants, however, engineering more significant genomic changes is more challenging due to reduced efficiency and precision. Here, we developed RecT Editor via Designer-Cas9-Initiated Targeting (REDIT), which leverages phage single-stranded DNA-annealing proteins (SSAP) RecT for mammalian genome engineering. Relative to Cas9-mediated homology-directed repair (HDR), REDIT yielded up to a 5-fold increase of efficiency to insert kilobase-scale exogenous sequences at defined genomic regions. We validated our REDIT approach using different formats and lengths of knock-in templates. We further demonstrated that REDIT tools using Cas9 nickase have efficient gene-editing activities and reduced off-target errors, measured using a combination of targeted sequencing, genome-wide indel, and insertion mapping assays. Our experiments inhibiting repair enzyme activities suggested that REDIT has the potential to overcome limitations of endogenous DNA repair steps. Finally, our REDIT method is applicable across cell types including human stem cells, and is generalizable to different Cas9 enzymes.

Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

The Herpes Simplex Viruses Utilize a Recombination‐Dependent Replication Mechanism to Replicate Viral Genomes

Although it has been known for decades that HSV replication results in the formation head‐to‐tail concatermers, the mechanism by which concatemers form is not understood. One long‐standing model involves circularization of incoming viral genomes followed by rolling circle replication. However, several lines of evidence suggest that HSV DNA replication may be more complex, relying instead on a recombination‐dependent replication mechanism. The HSV single‐strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a single strand annealing protein (SSAP). HSV encodes a conserved alkaline nuclease (UL12) that interacts with ICP8 to form a complex that is reminiscent of evolutionarily conserved phage recombinases, such as lambda red a/ß. We hypothesize that ICP8 and UL12 interact to promote recombination‐dependent DNA replication to generate head‐to‐tail concatemers necessary for the production of infectious virus. Due to the multifunctional nature of ICP8, it has been difficult to tea1se out the role of ICP8 as a SSAP from the various roles it plays during viral replication as a classical SSB. We have recently identified a novel ICP8 mutant, Q706A/F707A, that lacks annealing activity but retains many of the other functions characteristic of classical SSBs. Interestingly, this mutant is unable to synthesize viral DNA, indicating that the annealing function of ICP8 is essential for viral DNA synthesis (Weerasooriya, DiScipio et al. PNAS 2018)Annealing is a complex process requiring many types of molecular interactions. We hypothesize that it involves both ICP8‐DNA and ICP8‐ICP8 interactions as well as conformational changes induced by the recognition of complementary DNA. Based on their proximity to Q706A/F707A, we suggest that annealing may be mediated by a cluster of conserved residues (residues 701 to 719) in the shoulder domain of ICP8. We have identified five conserved arginine residues (R701, R704, R712, R713 and R714) in this region. Alanine mutagenesis of these residues showed that all mutants, except R704A are unable to complement the growth of a null mutant suggesting that these residues are important for viral growth. Biochemical characterization of purified mutant and WT proteins will be presented along with models for how ICP8 promotes efficient annealing during DNA synthesis.Support or Funding InformationNIH funding AI069136 and AI021747 to S.K.W and F30AI143125 to K.A.D

Crystal structure of the Redβ C-terminal domain in complex with λ Exonuclease reveals an unexpected homology with λ Orf and an interaction with Escherichia coli single stranded DNA binding protein.

Bacteriophage λ encodes a DNA recombination system that includes a 5′-3′ exonuclease (λ Exo) and a single strand annealing protein (Redβ). The two proteins form a complex that is thought to mediate loading of Redβ directly onto the single-stranded 3′-overhang generated by λ Exo. Here, we present a 2.3 Å crystal structure of the λ Exo trimer bound to three copies of the Redβ C-terminal domain (CTD). Mutation of residues at the hydrophobic core of the interface disrupts complex formation in vitro and impairs recombination in vivo. The Redβ CTD forms a three-helix bundle with unexpected structural homology to phage λ Orf, a protein that binds to E. coli single-stranded DNA binding protein (SSB) to function as a recombination mediator. Based on this relationship, we found that Redβ binds to full-length SSB, and to a peptide corresponding to its nine C-terminal residues, in an interaction that requires the CTD. These results suggest a dual role of the CTD, first in binding to λ Exo to facilitate loading of Redβ directly onto the initial single-stranded DNA (ssDNA) at a 3′-overhang, and second in binding to SSB to facilitate annealing of the overhang to SSB-coated ssDNA at the replication fork.

Herpes simplex virus 1 ICP8 mutant lacking annealing activity is deficient for viral DNA replication

Most DNA viruses that use recombination-dependent mechanisms to replicate their DNA encode a single-strand annealing protein (SSAP). The herpes simplex virus (HSV) single-strand DNA binding protein (SSB), ICP8, is the central player in all stages of DNA replication. ICP8 is a classical replicative SSB and interacts physically and/or functionally with the other viral replication proteins. Additionally, ICP8 can promote efficient annealing of complementary ssDNA and is thus considered to be a member of the SSAP family. The role of annealing during HSV infection has been difficult to assess in part, because it has not been possible to distinguish between the role of ICP8 as an SSAP from its role as a replicative SSB during viral replication. In this paper, we have characterized an ICP8 mutant, Q706A/F707A (QF), that lacks annealing activity but retains many other functions characteristic of replicative SSBs. Like WT ICP8, the QF mutant protein forms filaments in vitro, binds ssDNA cooperatively, and stimulates the activities of other replication proteins including the viral polymerase, helicase-primase complex, and the origin binding protein. Interestingly, the QF mutant does not complement an ICP8-null virus for viral growth, replication compartment formation, or DNA replication. Thus, we have been able to separate the activities of ICP8 as a replicative SSB from its annealing activity. Taken together, our data indicate that the annealing activity of ICP8 is essential for viral DNA replication in the context of infection and support the notion that HSV-1 uses recombination-dependent mechanisms during DNA replication.

Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB.

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

Domain Structure of the Redβ Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo

Redβ is a component of the Red recombination system of bacteriophage λ that promotes a single strand annealing (SSA) reaction to generate end-to-end concatemers of the phage genome for packaging. Redβ interacts with λ exonuclease (λexo), the other component of the Red system, to form a “synaptosome” complex that somehow integrates the end resection and annealing steps of the reaction. Previous work using limited proteolysis and chemical modification revealed that Redβ consists of an N-terminal DNA binding domain, residues 1–177, and a flexible C-terminal “tail”, residues 178–261. Here, we quantitatively compare the binding of the full-length protein (RedβFL) and the N-terminal domain (Redβ177) to different lengths of ssDNA substrate and annealed duplex product. We find that in general, RedβFL binds more tightly to annealed duplex product than to ssDNA substrate, while Redβ177 binds more tightly to ssDNA. In addition, the C-terminal region of Redβ corresponding to residues 182–261 was purified and found to fold into an α-helical domain that is required for the interaction with λexo to form the synaptosome complex. Deletion analysis of Redβ revealed that removal of just eleven residues from the C-terminus disrupts the interaction with λexo as well as ssDNA and dsDNA recombination in vivo. By contrast, the determinants for self-oligomerization of Redβ appear to reside solely within the N-terminal domain. The subtle but significant differences in the relative binding of RedβFL and Redβ177 to ssDNA substrate and annealed duplex product may be important for Redβ to function as a SSA protein in vivo.

Prophage recombinases-mediated genome engineering in Lactobacillus plantarum.

BackgroundLactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.ResultsIn this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum.ConclusionsThis Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0344-z) contains supplementary material, which is available to authorized users.

Destabilizing DNA during Rejoining Enhances Fidelity of Repair.

A new study shows that during repair of DNA, the effect of a single-strand annealing protein is to destabilize DNA duplex formation so that annealing only occurs between perfectly matched strands; the protein then clamps the strands together for repair. Read the Research Article.

A Single-Strand Annealing Protein Clamps DNA to Detect and Secure Homology.

Repair of DNA breaks by single-strand annealing (SSA) is a major mechanism for the maintenance of genomic integrity. SSA is promoted by proteins (single-strand-annealing proteins [SSAPs]), such as eukaryotic RAD52 and λ phage Redβ. These proteins use a short single-stranded region to find sequence identity and initiate homologous recombination. However, it is unclear how SSAPs detect homology and catalyze annealing. Using single-molecule experiments, we provide evidence that homology is recognized by Redβ monomers that weakly hold single DNA strands together. Once annealing begins, dimerization of Redβ clamps the double-stranded region and nucleates nucleoprotein filament growth. In this manner, DNA clamping ensures and secures a successful detection for DNA sequence homology. The clamp is characterized by a structural change of Redβ and a remarkable stability against force up to 200 pN. Our findings not only present a detailed explanation for SSAP action but also identify the DNA clamp as a very stable, noncovalent, DNA–protein interaction.

A new recombineering system for Photorhabdus and Xenorhabdus

Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.

Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases

Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×103 and (6.7±1.3)×103 streptomycin-resistant colonies per 108 viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5′ ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods.