Recombinant Acinetobacter sp. DNA polymerase IV (dinB)

What is DNA polymerase IV (dinB) and what is its fundamental role in bacterial DNA metabolism?

DNA polymerase IV (Pol IV) is a specialized Y-family DNA polymerase encoded by the dinB gene that plays crucial roles in translesion DNA synthesis (TLS) and mutagenesis. Unlike replicative polymerases, Pol IV exhibits no 3′→5′ exonuclease (proofreading) activity, making it inherently error-prone . In model organisms like E. coli, Pol IV is involved in non-targeted mutagenesis and is activated via SOS induction triggered by stalled polymerases at replication forks .

The primary function of Pol IV is to perform translesion synthesis across various DNA lesions that would otherwise block replicative polymerases. This ability allows bacteria to tolerate DNA damage and continue replication under stress conditions. Specifically, Pol IV can bypass certain types of DNA damage, including N2-deoxyguanine adducts, often at a faster rate than it traverses undamaged DNA .

While Acinetobacter sp. Pol IV shares fundamental characteristics with E. coli Pol IV, there may be species-specific adaptations in its function and regulation. The recombinant Acinetobacter sp. DNA polymerase IV protein consists of 351 amino acids , and like other bacterial Pol IV proteins, it likely plays important roles in DNA damage tolerance and potentially in the development of antibiotic resistance.

How is dinB expression regulated in bacterial systems?

The regulation of dinB expression varies across bacterial species but typically involves stress-responsive pathways. In the well-studied E. coli model:

• SOS Response Regulation: The dinB gene is under the transcriptional control of the LexA repressor and is induced in response to DNA damage as part of the SOS response .

• Expression Levels: Under normal conditions, approximately 250 molecules of Pol IV exist per E. coli cell, though more sensitive fluorescence microscopy measurements suggest this may be closer to 20 molecules per cell . During SOS induction, Pol IV production increases approximately 10-fold .

• Constitutive vs. Inducible Expression: Unlike E. coli, some bacterial species such as Mycobacterium tuberculosis exhibit constitutive expression of dinB homologs during both logarithmic growth and stationary phase, independent of RecA and the SOS response .

How do DinB proteins differ between bacterial species?

DNA polymerase IV exhibits notable differences across bacterial species in terms of structure, function, and regulation:

A significant difference is observed in mycobacterial DinB homologs, where DinB2 possesses robust RNA polymerase activity not typically seen in other bacterial Pol IV enzymes. This activity is determined by a "steric gate" residue (leucine instead of phenylalanine) that allows ribonucleotide incorporation . The mycobacterial DinB proteins also differ in their interaction with the β-clamp processivity factor, with only DinB1 showing this interaction .

Across species, these differences likely reflect adaptations to specific environmental niches and DNA damage profiles encountered by each bacterium.

What molecular mechanisms govern the Pol III-Pol IV switching during translesion synthesis?

The coordinated switching between the replicative Pol III and the specialized Pol IV during translesion synthesis involves a sophisticated molecular mechanism that ensures DNA damage tolerance while minimizing unnecessary mutagenesis:

Key Components of the Switching Mechanism:

• β Sliding Clamp Interactions: Pol IV interacts with both the rim and the cleft of the β sliding clamp. Mutations that disrupt either interaction (designated as Pol IV-R for rim mutations and Pol IV-C for cleft mutations) impair TLS function in vivo .

• Direct Pol III-Pol IV Interaction: Research has identified a direct interaction between Pol IV and Pol III that is essential for the polymerase switch. A specific mutation, Pol IV-T120P, abrogates this interaction while retaining full catalytic activity in vitro but failing to support TLS function in vivo .

• Regulatory Model: The switching mechanism appears to involve a three-component interaction where Pol IV must simultaneously engage with:

  • The β clamp rim

  • The β clamp cleft

  • The Pol III polymerase

This coordinated interaction allows Pol IV to displace Pol III from the primer terminus when necessary for lesion bypass, followed by a return to Pol III-mediated replication after the damage is bypassed .

Experimental Evidence:
Genetic selection studies have identified 13 novel Pol IV mutants that fail to impede E. coli growth when overproduced, suggesting they are defective in gaining access to the replication fork. Detailed biochemical analysis of these mutants confirms that the switching mechanism used under stress conditions is mechanistically similar to that employed under physiological conditions .

This switching mechanism represents a vitally important regulatory system that manages TLS in vivo by controlling Pol IV access to DNA, ensuring that specialized polymerases are deployed only when necessary.

How do structural features of dinB contribute to its substrate specificity and catalytic function?

The structural features of DNA polymerase IV dictate its unique substrate preferences, error patterns, and catalytic properties:

Critical Structural Elements:

• Steric Gate Residue: A key determinant of substrate selectivity is the "steric gate" residue, which prevents ribonucleotides from entering the active site. In E. coli DinB, this is phenylalanine 13 (F13) . Mycobacterial studies demonstrate that changing this residue from phenylalanine to leucine (F23L) or alanine (F23A) enables robust RNA polymerase activity .

• Active Site Flexibility: Unlike replicative polymerases, Pol IV has a more spacious active site that accommodates bulky DNA adducts, particularly at the N2 position of guanine. This flexibility comes at the cost of reduced fidelity on undamaged templates .

• Catalytic Domain: Pol IV is strictly distributive, meaning it incorporates only a few nucleotides before dissociating from the template. This property limits potential mutagenesis while allowing for targeted lesion bypass .

• Lack of Proofreading: The absence of 3'→5' exonuclease activity contributes to Pol IV's error-prone nature but is essential for its ability to bypass lesions that would otherwise trigger proofreading and stall replication .

Structure-Function Relationships:
Experimental evidence shows that Pol IV preferentially extends bulged (misaligned) primer/template structures, which explains its propensity for creating frameshift mutations . Site-directed mutagenesis experiments confirm that the polymerase activity of DinB is required for its in vivo mutagenicity .

The structural features that enable Pol IV to perform TLS also determine its lesion specificity. For instance, E. coli Pol IV efficiently bypasses N2-furfuryl-dG, N2-benzo[a]pyrene-dG, N2-carboxyethyl-dG, N2-N2-dG interstrand crosslinks, and various other N2-dG adducts , reflecting the specialized architecture of its active site.

What role does DNA polymerase IV play in antibiotic resistance development?

DNA polymerase IV contributes significantly to antibiotic resistance development through several mechanisms:

Mechanisms of Pol IV-Mediated Antibiotic Resistance:

• Ciprofloxacin Resistance: Studies demonstrate that Pol IV is a major determinant in the development of ciprofloxacin resistance in laboratory culture models. E. coli cells lacking Pol IV (ΔdinB) produce only 10% as many ciprofloxacin-resistant mutants as wild-type cells . This indicates Pol IV-dependent mutagenesis plays a crucial role in adapting to quinolone antibiotics.

• Adaptive Mutagenesis: Pol IV is involved in adaptive mutagenesis, a process where beneficial mutations arise in non-dividing or slowly dividing cells under stress conditions . This mechanism allows bacteria to develop resistance even in stationary phase or under growth-limiting antibiotic concentrations.

• Error-Prone Repair: Following DNA damage induced by antibiotics, Pol IV's error-prone nature during repair synthesis introduces mutations that may confer resistance. For example, in Acinetobacter baumannii, dinB appears to be involved in resistance acquisition mechanisms .

• Mutagenic Effects of Low-Fidelity Replication: Recent research shows that low-fidelity DNA polymerase IV can accelerate genome evolution. In one study, a Pol IV-deficient strain (mutT mutSβ dinB) exhibited a significant decrease in mutation rates compared to strains with active Pol IV .

Experimental Evidence:
Studies using complementation assays with arabinose-inducible plasmids containing wild-type or mutant dinB alleles confirm the direct involvement of Pol IV in increased mutagenesis. Expression of functional Pol IV from plasmid p5BAD-dinB increased mutation rates 2-fold relative to control cells, while cells carrying a plasmid encoding an inactive Pol IV mutant (p5BAD-dinBD8A) showed no enhancement of mutation rates .

These findings highlight the significant role of dinB in facilitating evolutionary adaptation to antibiotics, making it an important factor in the development of antibiotic resistance in various bacterial pathogens, including potentially Acinetobacter species.

How can researchers effectively design experiments to study dinB activity outside of replication contexts?

Recent evidence suggests that DNA polymerase IV operates significantly outside of DNA replication contexts, necessitating specialized experimental approaches:

Experimental Design Strategies:

• Single-Molecule Fluorescence Microscopy:

  • Technique: Construct fluorescently labeled dinB (e.g., dinB-YPet fusion) expressed from its natural promoter at its native chromosomal locus.

  • Application: Track the localization and dynamics of individual Pol IV molecules in living cells under various conditions.

  • Analysis: Quantify colocalization with replisomes (using markers like DnaX-mCherry) versus independent localization patterns.

  This approach has revealed that in E. coli, most Pol IV molecules carry out DNA synthesis predominantly outside replisomes, with only 5-10% of Pol IV foci colocalizing with replisome markers after DNA damage induction .

• Transcription-Associated Mutagenesis Assays:

  • Technique: Use reporter systems with inducible transcription and measure mutation frequencies in the presence or absence of functional dinB.

  • Application: Determine whether Pol IV contributes to mutagenesis during transcription, particularly at sites of transcription-replication conflicts.

• Recombination Intermediate Processing:

  • Technique: Employ in vitro reconstituted systems with purified recombination proteins (RecA, RecBCD) and DNA polymerase IV.

  • Application: Assess the ability of Pol IV to extend DNA synthesis from D-loop structures or other recombination intermediates.

• Time-Lapse DNA Damage Response Analysis:

  • Technique: Induce DNA damage with agents like ciprofloxacin, MMS, or UV light, then track changes in Pol IV concentrations and cellular locations through time using single-molecule tracking.

  • Results: This approach has shown that after ciprofloxacin treatment, E. coli cells exhibit stronger DinB-YPet signals with punctate foci visible, indicating binding to DNA damage sites .

Experimental Parameters Table:

By employing these specialized techniques, researchers can better understand the full spectrum of Pol IV activities in bacterial cells, including its roles in transcription, recombination, and double-strand break repair, which appear to be significant aspects of its cellular function beyond traditional TLS at replication forks.

What are the practical considerations for expression and purification of recombinant Acinetobacter sp. dinB protein?

The successful expression and purification of functional recombinant Acinetobacter sp. DNA polymerase IV requires careful consideration of several factors:

Expression System Considerations:

• Host Selection:

  • Bacterial Hosts: E. coli BL21(DE3) or derivatives are commonly used for expressing bacterial proteins.

  • Alternative Systems: Baculovirus expression in insect cells has been successfully used for Acinetobacter dinB expression, as noted in the product datasheet .

• Vector Design:

  • Promoter Selection: T7 or similar strong inducible promoters work well for controlled expression.

  • Affinity Tags: Consider N- or C-terminal tags (His6, GST, etc.) for purification, ensuring they don't interfere with activity.

  • Solubility Enhancement: Fusion partners like MBP or SUMO can improve solubility if needed.

Purification Protocol:

• Initial Extraction:

  • Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 200-300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, and protease inhibitors.

  • Sonication or alternative lysis methods followed by centrifugation to remove cell debris.

• Chromatography Steps:

  • Initial affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as a polishing step

• Storage Considerations:

  • The shelf life of liquid recombinant dinB is typically 6 months at -20°C/-80°C

  • The lyophilized form has an extended shelf life of 12 months at -20°C/-80°C

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

Quality Control Assessments:

• Purity Assessment: SDS-PAGE analysis to confirm >85% purity, as specified in the Acinetobacter dinB product datasheet .

• Activity Verification:

  • Primer extension assays using synthetic DNA templates

  • Divalent cation dependency tests with various metal ions

  • Lesion bypass assays if specific substrate preferences are being studied

• Protein Characterization:

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess proper folding

  • Thermal stability assays to determine optimal storage and reaction conditions

The Acinetobacter sp. DNA polymerase IV consists of 351 amino acids with the sequence provided in the product datasheet . Special attention should be given to ensuring proper folding and maintaining catalytic activity throughout the purification process.

How does the lesion bypass profile of Acinetobacter dinB compare to other bacterial homologs?

DNA polymerase IV from different bacterial species exhibits distinct lesion bypass profiles that reflect adaptations to their specific environmental challenges:

Comparative Lesion Bypass Profiles:

While specific data on Acinetobacter dinB lesion bypass profiles is limited in the provided search results, we can make some informed inferences based on the conservation of Y-family polymerase functions across bacteria:

• As a Y-family polymerase, Acinetobacter dinB likely specializes in bypassing certain types of bulky DNA adducts.

• Given that N2-dG adducts are commonly bypassed by bacterial dinB proteins, it's reasonable to hypothesize that Acinetobacter dinB would show activity on similar substrates.

• The absence of 3'→5' exonuclease activity, a common feature across dinB homologs, would likely make Acinetobacter dinB error-prone on undamaged DNA while allowing it to bypass lesions that would stall replicative polymerases.

To definitively establish the lesion bypass profile of Acinetobacter dinB, researchers would need to conduct in vitro bypass assays using purified recombinant protein with various damaged DNA templates, including those containing N2-dG adducts, oxidative lesions, and potentially environmental damage relevant to Acinetobacter's ecological niche.

What methodologies are recommended for studying dinB-mediated mutagenesis in vivo?

Investigating dinB-mediated mutagenesis in vivo requires specialized methodologies that can detect and characterize the specific mutational signatures:

Recommended Methodologies:

• Antibiotic Resistance Assays:

  • Procedure: Expose bacteria to sublethal concentrations of DNA-damaging agents, then plate on media containing antibiotics like rifampicin or ciprofloxacin.

  • Analysis: Compare mutation frequencies between wild-type and dinB-deficient strains.

  • Application: This approach revealed that E. coli cells lacking dinB produce only 10% as many ciprofloxacin-resistant mutants as wild-type cells .

• Fluctuation Analysis:

  • Procedure: Grow multiple parallel cultures from small inocula, then determine the number of mutants in each culture.

  • Analysis: Apply statistical methods (e.g., Luria-Delbrück distribution) to calculate mutation rates.

  • Advantage: Distinguishes between pre-existing mutations and those arising during the experiment.

• Reporter Assays:

  • Procedure: Utilize specialized reporter constructs (e.g., lacZ reversion assays, fluorescent proteins with inactivating mutations).

  • Analysis: Measure reversion frequencies or restoration of reporter function.

  • Application: Such assays have demonstrated Pol IV's role in adaptive mutagenesis in the E. coli lac operon .

• Whole Genome Sequencing:

  • Procedure: Compare whole genome sequences of parent strains and evolved mutants after exposure to stress conditions.

  • Analysis: Identify and catalog mutations to determine dinB-specific mutational signatures.

  • Advanced Application: Compare mutational profiles between wild-type, dinB-overexpressing, and dinB-deficient strains under various stresses.

• Genetic Complementation:

  • Procedure: Construct strains with inducible expression of wild-type or mutant dinB alleles.

  • Analysis: Measure restoration of mutagenesis in dinB-deficient backgrounds.

  • Example: Expression of functional Pol IV from plasmid p5BAD-dinB increased mutation rates 2-fold relative to control cells, while cells carrying a plasmid encoding an inactive Pol IV mutant showed no enhancement .

Experimental Design Considerations:

When designing experiments to study dinB-mediated mutagenesis in Acinetobacter or other bacteria, researchers should consider:

• DNA Damage Induction: Various DNA-damaging agents may induce different levels of dinB expression and activity. Options include:

  • Ciprofloxacin (DNA gyrase inhibitor that forms covalent adducts)

  • 4-nitroquinoline-1-oxide (NQO, forms bulky adducts)

  • Methylmethane sulfonate (MMS, alkylating agent)

  • UV light (forms pyrimidine dimers)

• Control Strains: Include appropriate controls such as:

  • Wild-type strain

  • dinB deletion strain

  • Strain expressing catalytically inactive dinB (e.g., D103N mutation)

  • Strains with mutations in other DNA repair pathways

• Mutational Spectrum Analysis: Beyond measuring mutation frequencies, characterize the types of mutations (transitions, transversions, frameshifts) to identify dinB-specific signatures.

These methodologies provide powerful tools for elucidating the role of dinB in mutagenesis and antibiotic resistance development in bacterial systems.

What are the implications of dinB's ability to incorporate ribonucleotides in certain bacterial species?

The discovery that some bacterial dinB homologs can efficiently incorporate ribonucleotides has significant implications for understanding bacterial physiology and evolution:

Biological Implications:

• Resource Utilization During Stress:

  • Bacteria in non-replicating states maintain much higher concentrations of rNTPs compared to dNTPs.

  • The ability to use ribonucleotides during DNA repair or mutagenesis could be advantageous under nutrient limitation or stress conditions .

• DNA Damage Control Strategy:

  • Incorporating ribonucleotides during gap repair or non-homologous end joining (NHEJ) may serve as a damage control strategy in non-replicating bacteria with limited dNTP pools .

  • These incorporated ribonucleotides could later be removed and replaced by deoxyribonucleotides when conditions improve.

• Evolutionary Implications:

  • The capacity to incorporate ribonucleotides may represent an ancestral function reflecting the proposed RNA world hypothesis.

  • It provides insights into the evolution of DNA polymerases from RNA polymerases.

Molecular Determinants:

The ability to incorporate ribonucleotides is determined by specific structural features:

• Steric Gate Residue:

  • In mycobacterial DinB proteins, the steric gate that typically prevents ribonucleotide incorporation varies between homologs.

  • DinB2 naturally contains a leucine instead of phenylalanine at this position, enabling efficient RNA polymerase activity .

  • Experimental evidence shows that changing DinB1's steric gate Phe23 to leucine (F23L) or alanine (F23A) confers RNA polymerase activity .

• Template Preferences:

  • Studies with mycobacterial DinB2 showed it has:

    • Strong DNA-dependent RNA polymerase activity

    • Good RNA-dependent DNA polymerase (reverse transcriptase) activity

    • Limited RNA-dependent RNA polymerase activity

Potential Research Applications:

• Novel Biotechnological Tools:

  • DinB variants with RNA polymerase activity could be developed as specialized tools for RNA-related applications.

  • Their unique properties might be useful for methods requiring both DNA and RNA synthesis capabilities.

• Therapeutic Targets:

  • Understanding ribonucleotide incorporation by bacterial DinB proteins could lead to novel antibacterial strategies targeting this unusual activity.

  • Inhibitors specific to this function might selectively target bacteria that rely on this activity during infection.

• Evolutionary Studies:

  • These polymerases provide natural systems to study the transition between RNA and DNA worlds.

  • They offer insights into how nature solves the problem of template-dependent nucleic acid synthesis with different substrates.

While there is no specific information about ribonucleotide incorporation by Acinetobacter dinB in the provided search results, this property could be experimentally investigated using approaches similar to those used with mycobacterial homologs, including site-directed mutagenesis of the putative steric gate residue and assays with various template-primer configurations.

What are the most promising future research directions for Acinetobacter sp. DNA polymerase IV studies?

Based on current knowledge gaps and the importance of dinB in bacterial physiology and adaptation, several promising research directions emerge:

• Structure-Function Relationship:

  • Determine the crystal structure of Acinetobacter dinB to understand its specific structural features.

  • Conduct comparative structural analysis with other bacterial homologs to identify unique elements.

  • Use site-directed mutagenesis to investigate key residues controlling substrate specificity and catalytic activity.

• Role in Antibiotic Resistance:

  • Investigate whether dinB contributes to the notorious antibiotic resistance capabilities of Acinetobacter species, particularly A. baumannii.

  • Determine if dinB overexpression occurs during antibiotic exposure in clinical isolates.

  • Develop strategies to modulate dinB activity as potential adjuvants to antibiotic therapy.

• Environmental Adaptation:

  • Explore whether Acinetobacter dinB contributes to adaptation to diverse environmental niches.

  • Investigate dinB's role in stress responses unique to Acinetobacter species.

  • Compare dinB function across environmental and clinical Acinetobacter isolates to identify potential differences.

• Specialized Functions:

  • Determine whether Acinetobacter dinB possesses ribonucleotide incorporation activity similar to mycobacterial homologs.

  • Investigate potential roles in recombination, transcription, or other DNA metabolic processes beyond traditional TLS.

  • Explore interactions with other components of DNA replication and repair machinery in Acinetobacter.

• Biotechnological Applications:

  • Develop Acinetobacter dinB as a biotechnological tool for specialized DNA synthesis applications.

  • Engineer variants with enhanced capabilities for specific research or diagnostic applications.

  • Explore its potential use in techniques requiring specialized polymerase activities.

The study of Acinetobacter sp. DNA polymerase IV not only contributes to our fundamental understanding of bacterial DNA damage tolerance but may also provide insights into mechanisms of antibiotic resistance and environmental adaptation in this important bacterial genus, which includes significant opportunistic pathogens.

How can contradictory findings in dinB research be reconciled through improved experimental design?

Several contradictions exist in the literature regarding dinB function and its effects on mutation rates. These can be addressed through careful experimental design:

Common Contradictions and Reconciliation Approaches:

• Mutagenic vs. Accurate TLS:

  • Contradiction: Some studies show dinB increases mutation rates, while others suggest it accurately bypasses certain lesions like 8-oxoguanine .

  • Reconciliation: Design experiments that test mutagenicity on different lesion types under identical conditions. The template sequence context and lesion type likely determine whether bypassing is accurate or mutagenic.

• Expression Levels:

  • Contradiction: Early Western blot studies estimated ~250 molecules of Pol IV per E. coli cell, while fluorescence microscopy suggests closer to 20 molecules per cell .

  • Reconciliation: Use multiple independent methods (Western blotting, fluorescence microscopy, mass spectrometry) on the same strains under identical conditions, with appropriate controls and calibration curves.

• Replisome Association:

  • Contradiction: The traditional model suggests Pol IV acts within replisomes, while recent fluorescence microscopy indicates most Pol IV molecules function outside replisomes .

  • Reconciliation: Combine single-molecule tracking with biochemical approaches and genetic studies, examining different damage types and growth conditions.

• Role in Antibiotic Resistance:

  • Contradiction: The contribution of dinB to antibiotic resistance varies across studies and bacterial species.

  • Reconciliation: Standardize experimental conditions, use multiple antibiotics, and consider strain background effects. Sequence dinB genes from multiple isolates to identify potential polymorphisms affecting function.

Methodological Improvements:

• Standardization of Assay Conditions:

  • Use consistent growth conditions, media compositions, and damage induction protocols.

  • Report detailed experimental parameters to enable replication.

• Multi-Method Validation:

  • Apply complementary approaches (genetic, biochemical, structural, and imaging) to the same biological questions.

  • Confirm key findings using independent methodologies.

• Physiologically Relevant Conditions:

  • Study dinB function under conditions that mimic natural bacterial environments.

  • Consider growth phase, nutrient availability, and relevant stressors.

• Advanced Statistical Analysis:

  • Apply rigorous statistical methods appropriate for the data type.

  • Consider potential biases and confounding factors in experimental design.

• Controls for Protein Expression Levels:

  • Carefully control and quantify protein expression levels in complementation studies.

  • Use titrated expression systems rather than simple presence/absence comparisons.