Recombinant Acinetobacter sp. DNA replication and repair protein recF (recF)

What is RecF and what is its primary function in bacterial cells?

RecF is a DNA-binding protein involved in the RecFOR pathway of DNA repair, which is critical for maintaining genome stability during replication and for repairing DNA damage. RecF functions as part of a complex with RecO and RecR proteins to facilitate the loading of RecA onto DNA at stalled replication forks, promoting fork stability and the eventual resumption of replication. RecF specifically directs RecO and RecR to DNA junctions generated at blocked replication forks and mediates the loading and formation of RecA nucleoprotein filaments at these sites . The protein operates as a member of the Structural Maintenance of Chromosome (SMC) proteins and ATP Binding Cassette (ABC) ATPases family, utilizing ATP hydrolysis for its functions in DNA binding and repair processes .

How is RecF structurally characterized?

The crystal structure of RecF (as determined in Deinococcus radiodurans) reveals that it forms a homo-dimeric complex with a distinctive clam-like structure containing an ABC ATPase domain . The protein contains three highly conserved motifs essential for its function:

• Walker A motif - Located in ATPase domain 1 at the N-terminus

• Walker B motif - Located in ATPase domain 1 at the C-terminus

• Signature motif - Residing in domain 2

These structural elements are characteristic of ABC ATPases and enable RecF to bind and hydrolyze ATP during DNA repair processes. The dimeric structure allows RecF to potentially bridge DNA structures at replication forks, facilitating repair complex assembly.

What phenotypes are observed in RecF-deficient bacterial strains?

RecF-deficient strains exhibit several distinctive phenotypes that illuminate the protein's function:

• Hypersensitivity to UV irradiation, particularly when cells are actively replicating at the time of damage

• Extensive degradation of nascent DNA strands (estimated up to 20 kb) following UV-induced DNA damage

• Failure to maintain replication fork integrity after encountering DNA damage

• Impaired ability to resume DNA synthesis following disruption

• Accumulation of persistent gaps in nascent DNA

• High rates of DNA strand exchanges

Importantly, these phenotypes are most pronounced when DNA damage occurs during active replication, suggesting RecF's primary role is in processing DNA damage encountered by replication forks rather than in processing DNA damage itself .

How does RecF cooperate with other proteins in DNA repair pathways?

RecF operates within a coordinated network of proteins in the RecFOR pathway:

In this pathway, RecF, RecO, and RecR work together to displace SSB proteins and enhance RecA nucleoprotein filament formation at arrested replication forks . Subsequently, RecQ and RecJ act in concert to process the nascent DNA at the replication fork, exposing the DNA region containing damage. This processing is thought to restore the lesion-containing region to a double-stranded substrate that can be acted upon by nucleotide excision repair .

What methodological approaches are used to express and purify recombinant Acinetobacter RecF?

Expression and purification of recombinant Acinetobacter RecF requires several methodological considerations:

• Expression system selection: E. coli BL21(DE3) or similar strains are typically used with pET-based vectors containing optimized codons for Acinetobacter genes.

• Expression optimization: Since RecF is an ATP-binding protein that forms dimers, expression conditions must be carefully optimized:

  • Lower induction temperatures (16-20°C) often improve proper folding

  • IPTG concentration typically between 0.1-0.5 mM

  • Extended expression time (overnight) at lower temperatures

• Purification strategy:

  • Initial capture typically employs metal affinity chromatography (IMAC) using His-tagged constructs

  • Ion exchange chromatography to separate ATP-bound and free forms

  • Size exclusion chromatography to isolate properly folded dimeric species and remove aggregates

  • Typical yields range from 2-5 mg/L of bacterial culture

• Functional verification:

  • ATP binding and hydrolysis assays

  • DNA binding assays with forked DNA substrates

  • Interaction studies with RecO and RecR proteins

The purified protein must be maintained in buffers containing glycerol (10-20%) and reducing agents (1-5 mM DTT or β-mercaptoethanol) to preserve activity during storage.

How can structure-function relationships in RecF be analyzed through directed mutagenesis?

Structure-function analysis of RecF can be systematically approached through targeted mutations:

• ATP binding and hydrolysis mutations:

  • Walker A motif (typically K→A substitution): Disrupts ATP binding

  • Walker B motif (typically D→N substitution): Allows ATP binding but prevents hydrolysis

  • These mutations help determine whether ATP binding alone or ATP hydrolysis is required for specific RecF functions

• DNA binding interface mutations:

  • Positively charged residues (K, R) in the DNA binding domains can be mutated to neutral or negatively charged residues

  • These mutations differentiate activities that require DNA binding from those that don't

• Dimerization interface mutations:

  • Alter residues at the dimer interface to create monomeric variants

  • These help determine whether dimerization is essential for all RecF functions or only subset activities

As noted in search result , the goal of these mutations is to "identify mutations that may arrest the recovery process at unique stages, thereby illuminating the steps that these proteins catalyze or participate in during the processing of lesions encountered during replication" . Functional assays with these mutants should include DNA binding, ATP hydrolysis, RecA loading capability, and in vivo complementation testing.

What specialized assays can be used to analyze RecF activity and its role in replication fork maintenance?

Several specialized assays provide insights into RecF activity:

• DNA binding assays:

  • Electrophoretic mobility shift assays (EMSA) with various DNA structures

  • Fluorescence anisotropy with labeled DNA substrates

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• ATPase activity assays:

  • Colorimetric assays measuring phosphate release

  • Coupled enzyme assays with pyruvate kinase/lactate dehydrogenase

  • Radioactive [γ-32P]ATP hydrolysis assays for enhanced sensitivity

• Replication fork recovery assays:

  • DNA synthesis measurement following UV damage using pulse-labeling techniques

  • Nascent DNA degradation assays to quantify processing at stalled forks

  • Replication fork regression and restoration assays using purified components

• In vivo functional complementation:

  • UV survival assays with RecF-deficient strains complemented with wild-type or mutant RecF

  • DNA replication restart assays following DNA damage

  • Measurement of RecA-GFP focus formation in cells with various RecF alleles

These assays together can dissect the molecular mechanism by which RecF facilitates replication fork maintenance and repair, particularly in the context of Acinetobacter species' unique genome organization and repair mechanisms.

How might RecF function intersect with plasmid-based recombination in Acinetobacter species?

In Acinetobacter species, RecF function may have significant implications for plasmid stability and recombination:

• Plasmid stability during replication stress:

  • RecF's role in maintaining stalled replication forks likely extends to plasmid replication

  • In Acinetobacter, plasmids containing XerC/D sites undergo reversible remodeling through site-specific recombination

  • RecF may influence this process by stabilizing replication forks on plasmids during recombination events

• Interaction with plasmid recombination machinery:

  • RecF could potentially interact with or influence the XerC/D-mediated recombination system common in Acinetobacter plasmids

  • The reversible remodeling of Acinetobacter plasmid structures mediated by different pairs of pXerC/D sites impacts host adaptation to challenging environments

  • RecF may modulate these recombination frequencies through its replication fork maintenance activities

• Experimental approaches to study this intersection:

  • Transformation efficiency assays with recombination-prone plasmids in RecF+ vs. RecF- backgrounds

  • Analysis of plasmid structural diversity in populations with different RecF status

  • In vitro reconstitution of RecF and XerC/D-mediated recombination on model substrates

  • PCR-based detection methods similar to those used in Acinetobacter plasmid studies

This intersection is particularly relevant since Acinetobacter species harbor clinically important plasmids carrying antibiotic resistance genes, such as the blaOXA-58 carbapenemase gene mentioned in the research .

What are the current challenges and future directions in RecF research in Acinetobacter species?

Several challenges and future directions exist in RecF research:

• Technical challenges:

  • Establishing genetic manipulation systems in clinical Acinetobacter isolates

  • Creating clean deletion mutants in strains with redundant DNA repair pathways

  • Purifying sufficient quantities of active recombinant protein from Acinetobacter species

  • Developing in vitro systems that accurately recapitulate the complexity of replication fork collapse and recovery

• Knowledge gaps:

  • Species-specific variations in RecF function between different Acinetobacter species

  • The interplay between RecF and mobile genetic elements during horizontal gene transfer

  • The impact of RecF on antibiotic resistance acquisition and evolution

  • How environmental stressors modulate RecF activity in Acinetobacter species

• Future research directions:

  • Single-molecule studies to visualize RecF dynamics at replication forks

  • Cryo-EM structures of RecF-RecO-RecR complexes on DNA substrates

  • Genome-wide mapping of RecF binding sites under various stress conditions

  • Development of RecF inhibitors as potential antibiotic adjuvants to limit evolution of resistance

• Methodological innovations needed:

  • Systems for controlled expression of RecF variants in Acinetobacter

  • High-throughput screening methods to identify RecF modulators

  • Improved in vitro reconstitution of complete replication fork recovery

As noted in the search results, further work is needed to understand "the influences of the different Acinetobacter pXerC/D core sequences and genetic context on the feasibility and directionality of the recombination reaction" , and similar detailed mechanistic studies are needed for RecF function in this important bacterial genus.

How should experiments be designed to distinguish RecF-specific effects from those of other RecFOR pathway proteins?

Designing experiments to isolate RecF-specific effects requires several strategic approaches:

• Genetic separation of functions:

  • Create single, double, and triple deletion mutants of recF, recO, and recR

  • Complement with wild-type or mutant alleles of each gene individually

  • Use depletion systems (e.g., CRISPR interference or degron tags) for temporal control

• Biochemical separation:

  • Perform in vitro reconstitution experiments with various combinations of purified RecF, RecO, and RecR proteins

  • Use order-of-addition experiments to determine the sequence of events

  • Employ RecF mutants that retain specific functions (e.g., DNA binding but not ATP hydrolysis)

• Structural and interaction analysis:

  • Map specific interaction interfaces between RecF and other pathway proteins

  • Create separation-of-function mutants that disrupt specific protein-protein interactions

  • Use crosslinking approaches to capture transient complexes

• Temporal analysis:

  • Employ synchronized cell populations to examine RecF recruitment to stalled forks

  • Use real-time single-molecule approaches to track the order of protein assembly

These approaches can help determine which phenotypes result directly from RecF activity versus those that require the complete RecFOR complex.

How does RecF structure and function in Acinetobacter compare to other bacterial species?

RecF is highly conserved across bacterial species, but exhibits some important variations:

How does the RecFOR pathway interact with other DNA repair mechanisms in Acinetobacter?

The RecFOR pathway interfaces with multiple DNA repair mechanisms:

• Nucleotide Excision Repair (NER):

  • RecFOR pathway and NER show synergistic enhancement of survival, implying they function in a common pathway to promote cell survival

  • After RecQ and RecJ process the stalled replication fork, NER (UvrABC) can access and remove lesions in the exposed DNA

  • This coordinated action allows for efficient repair and resumption of replication

• Translesion Synthesis (TLS):

  • When lesions cannot be removed by NER, alternative TLS processes can occur

  • These allow DNA synthesis to resume with lower efficiency and higher mutation frequency

  • RecF likely influences the balance between high-fidelity repair and error-prone TLS

• Homologous Recombination:

  • RecF facilitates RecA loading, which is essential for homologous recombination

  • This creates a direct mechanistic link between replication fork maintenance and recombinational repair

• Site-Specific Recombination:

  • In Acinetobacter, XerC/D-mediated site-specific recombination contributes to genomic plasticity

  • RecF may influence how these recombination events proceed at replication forks

Understanding these pathway interactions is crucial for developing comprehensive models of DNA repair in Acinetobacter species and potentially identifying targets for combination therapies against drug-resistant strains.

What are the implications of RecF research for understanding antimicrobial resistance evolution in Acinetobacter?

RecF research has significant implications for antimicrobial resistance (AMR) evolution:

• Genomic plasticity mechanisms:

  • RecF's role in maintaining replication forks influences how bacteria can tolerate DNA damage and mutation

  • This directly impacts the acquisition and maintenance of resistance mutations

  • In Acinetobacter, this may be particularly important due to the heavy burden of mobile genetic elements and resistance genes

• Plasmid stability and transfer:

  • RecF likely influences the stability of resistance plasmids during replication stress

  • As noted in the research, Acinetobacter plasmids carrying carbapenem resistance genes like blaOXA-58 undergo dynamic structural changes

  • RecF may affect how these plasmids are maintained and transferred between bacteria

• Stress-induced mutagenesis:

  • RecF's function at stalled replication forks influences how cells respond to antibiotic stress

  • This response pathway can potentially modulate mutation rates under selective pressure

• Potential therapeutic targets:

  • Understanding RecF function could identify targets to limit evolution of resistance

  • Inhibitors of RecF might serve as adjuvants to conventional antibiotics

  • Combination approaches targeting both bacterial survival and adaptation mechanisms could provide more sustainable antimicrobial strategies