Recombinant Thermotoga maritima DNA topoisomerase 1 (topA), partial

What is Thermotoga maritima DNA topoisomerase I and what is its functional significance?

Thermotoga maritima DNA topoisomerase I (topA) is a type I topoisomerase found in the hyperthermophilic bacterium T. maritima. Like other bacterial topoisomerases I, it participates in the management of DNA topology during replication, transcription, and recombination by controlling DNA supercoiling. The enzyme belongs to the bacterial type IA topoisomerase family, catalyzing the relaxation of negatively supercoiled DNA through a strand passage mechanism involving transient single-strand breaks .

T. maritima is particularly interesting among bacteria because it contains both reverse gyrase (typically found in hyperthermophiles) and DNA gyrase, which introduces negative supercoiling in DNA. This unique combination raises important questions about DNA topology management at extremely high temperatures . The presence of these complementary topoisomerases may provide T. maritima with more sophisticated control over DNA topology than organisms possessing only one type.

What are the structural domains of T. maritima DNA topoisomerase I and how do they contribute to function?

T. maritima DNA topoisomerase I consists of two primary structural domains with distinct functions:

• Core Domain (TmTop65): Contains all the conserved motifs involved in trans-esterification reactions necessary for DNA cleavage and rejoining. This domain harbors the catalytic site responsible for the basic topoisomerase activity. Notably, unlike its E. coli counterpart, the T. maritima core domain can sustain a complete topoisomerization cycle independently, though with reduced efficiency .

• Carboxyl-Terminal Domain: Highly variable in size and sequence among different bacterial species. While not strictly required for the strand passage reaction, this domain strongly influences DNA binding efficiency and cleavage specificity . When the T. maritima core domain is fused with the E. coli carboxyl-terminal domain, the resulting chimera shows considerably increased binding efficiency, thermal stability, and DNA relaxation activity.

The functional interplay between these domains enables the enzyme to efficiently bind DNA, introduce single-strand breaks, facilitate strand passage, and religate the DNA strand to alter DNA topology.

What expression systems and purification strategies are most effective for recombinant T. maritima DNA topoisomerase I?

While the search results don't provide specific expression protocols for T. maritima topoisomerase I, effective strategies can be inferred from related research on thermophilic proteins:

Expression Systems:

• E. coli BL21(DE3) or similar strains with T7 RNA polymerase-based expression vectors are typically used for thermophilic proteins

• Temperature-inducible or IPTG-inducible promoters allow controlled expression

• Co-expression with chaperones may improve folding of thermophilic proteins in mesophilic hosts

• Expression at moderately elevated temperatures (30-37°C) often yields better results than standard conditions

Purification Strategies:

• Heat treatment (70-80°C) as an initial purification step leverages the thermostability of T. maritima proteins to remove heat-labile E. coli proteins

• Affinity chromatography using His-tagged constructs or specialized columns (similar to the novobiocin-Sepharose column used for T. maritima DNA gyrase)

• Ion exchange chromatography to separate based on charge properties

• Size exclusion chromatography as a polishing step

To verify enzyme activity, DNA relaxation assays using negatively supercoiled plasmid DNA should be performed at elevated temperatures (80-85°C) to match the enzyme's thermophilic nature.

What are the optimal reaction conditions for T. maritima DNA topoisomerase I activity?

Based on the search results and the hyperthermophilic nature of T. maritima, the optimal reaction conditions for topoisomerase I activity likely include:

Temperature: DNA gyrase from T. maritima has optimal activity around 82-86°C , suggesting that topoisomerase I would function optimally in a similar temperature range, consistent with the organism's growth temperature.

pH: While specific information for T. maritima topoisomerase I pH optimum is not provided, other T. maritima enzymes like SurE show optimal activity at pH 5.5-6.2 . Topoisomerase I likely functions best in a slightly acidic to neutral pH range.

Salt Concentration: Moderate monovalent cation concentrations (likely 50-150 mM NaCl or KCl) would stabilize DNA-protein interactions without interfering with catalytic activity.

Divalent Cations: Mg²⁺ is essential for type IA topoisomerase activity, typically at 5-10 mM concentration, as it coordinates the phosphodiester bond cleavage and religation reactions.

Reducing Agents: DTT or β-mercaptoethanol (1-5 mM) to maintain cysteine residues in a reduced state for optimal activity.

Optimizing these conditions is critical for experimental reproducibility when working with this thermophilic enzyme.

How does the partial recombinant T. maritima DNA topoisomerase I compare to the full-length enzyme?

The partial recombinant T. maritima DNA topoisomerase I (referring to the core domain, TmTop65) shows distinct functional differences compared to the full-length enzyme:

• Catalytic Activity: Unlike the E. coli core domain, TmTop65 can perform a complete topoisomerization cycle independently, though with significantly reduced efficiency compared to the full-length enzyme . This suggests the core domain contains all essential catalytic elements.

• DNA Binding: The partial enzyme exhibits substantially reduced DNA binding efficiency, indicating that the carboxyl-terminal domain significantly contributes to substrate recognition and binding stability .

• Thermal Stability: While both versions are thermostable, fusion of TmTop65 to the E. coli carboxyl-terminal domain increases thermal stability, suggesting the C-terminal domain enhances structural integrity .

• Cleavage Specificity: The partial and full-length enzymes show different DNA cleavage site preferences, demonstrating that the C-terminal domain influences substrate positioning within the active site .

These differences highlight the complementary roles of both domains in achieving optimal enzymatic function while maintaining the fundamental catalytic capability within the core domain.

How does the core domain (TmTop65) of T. maritima DNA topoisomerase I function without the carboxyl-terminal domain?

The core domain of T. maritima topoisomerase I (TmTop65) demonstrates a remarkable functional independence not observed in mesophilic counterparts like E. coli topoisomerase I. Research indicates that TmTop65 can sustain a complete topoisomerization cycle by itself, although with reduced efficiency . This self-sufficiency involves several mechanistic aspects:

• Minimal DNA Binding: TmTop65 retains sufficient DNA binding capability to engage with DNA substrates, though with much lower affinity than the full-length enzyme. This contrasts with E. coli topoisomerase I, where the core domain alone cannot bind DNA efficiently enough to complete the reaction cycle .

• Complete Catalytic Machinery: The core domain contains all essential catalytic residues for DNA cleavage, strand passage, and religation reactions. The active site architecture remains functional even without the contribution of the C-terminal domain .

• Altered Reaction Kinetics: While capable of completing the topoisomerization cycle, TmTop65 likely exhibits reduced processivity (fewer topological changes per binding event) and altered reaction rates due to the compromised DNA binding stability.

• Evolutionary Implications: This functional independence suggests that the core domain represents the ancestral minimal functional unit for topoisomerization, with the C-terminal domain evolving later to enhance efficiency and specificity rather than enable basic catalytic function.

This functional self-sufficiency may represent an adaptation to extreme environments, providing a more robust enzymatic mechanism that maintains minimal activity even under conditions that might compromise domain-domain interactions.

What structural features contribute to the thermostability of T. maritima DNA topoisomerase I?

T. maritima DNA topoisomerase I must maintain structural integrity and catalytic function at extremely high temperatures (optimal around 80-85°C). While the search results don't provide explicit details about the thermostability features of this specific enzyme, information about other T. maritima proteins and general principles of protein thermostability in hyperthermophiles suggest several key structural adaptations:

• Enhanced Ionic Networks: T. maritima proteins typically contain extensive intra- and inter-subunit salt bridges that strengthen at high temperatures and contribute significantly to thermostability, as identified in the T. maritima SurE protein . These electrostatic networks likely stabilize the tertiary structure of topoisomerase I.

• Compacted Hydrophobic Core: Thermostable proteins often feature tighter packing of hydrophobic residues in their core, reducing cavities and enhancing van der Waals interactions that resist thermal denaturation.

• Increased Secondary Structure Content: Higher proportion of α-helices and β-sheets with reduced loop regions limits conformational flexibility and entropy-driven unfolding at elevated temperatures.

• Surface Charge Optimization: Strategically positioned charged residues on the protein surface can form stabilizing ionic interactions that become increasingly favorable at high temperatures.

• Metal Ion Coordination: Additional metal-binding sites may contribute to maintaining structural integrity under extreme conditions.

Understanding these thermostability features has significant implications for protein engineering efforts aimed at creating heat-resistant enzymes for biotechnological applications.

How do chimeric constructs of T. maritima and E. coli topoisomerase I domains affect DNA binding specificity and activity?

Studies involving chimeric constructs of T. maritima and E. coli topoisomerase I domains have revealed critical insights into domain functions and interdependencies. Two key chimeric constructs show particularly interesting properties:

• TmTop65-EcCTD Chimera (T. maritima core + E. coli C-terminal domain):

  • Demonstrates considerably increased DNA binding efficiency compared to TmTop65 alone

  • Shows enhanced thermal stability over the T. maritima core domain

  • Exhibits significantly improved DNA relaxation activity

  • Most notably, predominantly acquires the cleavage site specificity characteristic of E. coli topoisomerase I

• EcTop67-TmCTD Chimera (E. coli core + T. maritima C-terminal domain):

  • Shows very poor DNA relaxation activity

  • Exhibits weak DNA binding capability

  • Formation of covalent DNA adducts is severely impaired

  • Suggests fundamental incompatibility between these particular domains

These findings demonstrate that the carboxyl-terminal domain strongly determines DNA binding efficiency and dictates cleavage site specificity. The dramatic difference in functionality between the two chimeras suggests that despite evolutionary conservation of the basic topoisomerase mechanism, the interface between core and C-terminal domains has evolved differently in thermophilic versus mesophilic bacteria.

This domain-swapping approach provides valuable insights for protein engineering efforts aimed at creating topoisomerases with novel properties combining thermostability with specific DNA interaction characteristics.

What experimental approaches can resolve contradictory findings regarding DNA supercoiling in hyperthermophiles?

The search results reveal an important contradiction: while plasmids from hyperthermophilic archaea range from relaxed to positively supercoiled, the plasmid pRQ7 from Thermotoga sp. is negatively supercoiled . This challenges previous assumptions about DNA topology in hyperthermophiles and requires sophisticated experimental approaches to resolve:

• Temperature-Controlled Topological Analysis:

  • Design in vivo experiments using rapid quenching techniques to capture DNA topology at different temperatures

  • Employ two-dimensional gel electrophoresis with chloroquine to visualize the distribution of topoisomers

  • Conduct time-course experiments following temperature shifts to capture dynamic changes in supercoiling

• Genetic Manipulation Studies:

  • Create conditional knockdowns or temperature-sensitive mutants of reverse gyrase and DNA gyrase in T. maritima

  • Monitor the effects on DNA topology and cell viability at different temperatures

  • Perform complementation studies with topoisomerases from different thermophilic origins

• Biochemical Characterization:

  • Quantify the relative activities and expression levels of reverse gyrase versus DNA gyrase at different temperatures

  • Examine the response of these enzymes to physiologically relevant molecular crowding conditions

  • Investigate potential direct or indirect regulatory interactions between these topoisomerases

• Comparative Genomics:

  • Analyze the distribution of topoisomerase genes across hyperthermophilic species

  • Correlate topoisomerase complements with optimal growth temperatures and genomic features

  • Identify potential accessory proteins that might modulate topoisomerase activity in different species

These approaches could determine whether the contradictory findings represent fundamentally different strategies for DNA management between hyperthermophilic bacteria and archaea, or whether they reflect a more complex, temperature-dependent regulation of DNA topology.

How does the presence of both reverse gyrase and DNA gyrase in T. maritima affect DNA topology at high temperatures?

The coexistence of both reverse gyrase and DNA gyrase in T. maritima represents a unique topological management system among hyperthermophiles. This dual topoisomerase system has significant implications for DNA topology regulation:

• Opposing Topological Activities: Reverse gyrase introduces positive supercoils into DNA (typically associated with hyperthermophiles), while DNA gyrase introduces negative supercoils. This counterintuitive combination suggests a sophisticated regulatory system for dynamic control of DNA topology .

• Experimental Evidence: The plasmid pRQ7 from Thermotoga sp. maintains negative supercoiling under normal growth conditions and only becomes positively supercoiled after inhibition of DNA gyrase with novobiocin. This confirms that negative supercoiling in Thermotoga is actively maintained by DNA gyrase activity despite the presence of reverse gyrase .

• Temperature-Dependent Regulation: At extremely high temperatures (optimal growth ~80°C), the interplay between these enzymes likely enables fine-tuned control of DNA topology. DNA gyrase may counteract excessive positive supercoiling that would otherwise accumulate due to thermal effects and reverse gyrase activity.

• Functional Implications: This balanced system may facilitate more efficient DNA transactions (replication, transcription, recombination) by maintaining optimal topology across varying temperature conditions and cellular states.

• Evolutionary Significance: As noted in the research, "The findings concerning DNA gyrase and negative supercoiling in Thermotogales put into question the role of reverse gyrase in hyperthermophiles" . This suggests that previous assumptions about the necessity of positive supercoiling for DNA stability at high temperatures require reconsideration.

This dual topoisomerase system likely represents a sophisticated evolutionary adaptation providing T. maritima with more flexible control over DNA topology than organisms possessing only one type of supercoiling activity.

What mechanistic differences exist between T. maritima topoisomerase I and mesophilic bacterial topoisomerases?

Several significant mechanistic differences distinguish T. maritima topoisomerase I from its mesophilic counterparts like E. coli topoisomerase I:

• Core Domain Functionality: The core domain of T. maritima topoisomerase I (TmTop65) can sustain a complete topoisomerization cycle independently, albeit with low efficiency. In contrast, the core domain of E. coli topoisomerase I cannot function without its C-terminal domain . This suggests a more self-sufficient catalytic mechanism in the thermophilic enzyme.

• Thermostability Mechanisms: T. maritima topoisomerase I employs specialized structural features (likely including extensive salt bridge networks similar to those identified in other T. maritima proteins ) to maintain structural integrity and activity at temperatures that would denature mesophilic enzymes.

• Temperature Optimum: T. maritima topoisomerase I functions optimally at extremely high temperatures (likely 80-90°C based on the optimal temperature of T. maritima DNA gyrase at 82-86°C ), whereas mesophilic topoisomerases typically function optimally around 37°C.

• DNA Substrate Interaction: The chimeric construct studies revealed differences in how the enzymes interact with DNA substrates, as evidenced by the observation that chimeric constructs acquire the cleavage specificity of the enzyme contributing the C-terminal domain .

• Cellular Context: In T. maritima, topoisomerase I operates in an environment containing both reverse gyrase and DNA gyrase , a unique situation not found in mesophilic bacteria, suggesting potential functional interplay between these enzymes in managing DNA topology at high temperatures.

These mechanistic differences reflect evolutionary adaptations to distinct environmental niches and demonstrate how homologous enzymes can develop specialized mechanisms while maintaining the same fundamental catalytic function.

How can recombinant T. maritima DNA topoisomerase I be engineered for enhanced properties or novel applications?

Based on the understanding of T. maritima topoisomerase I structure-function relationships, several rational engineering approaches could enhance its properties for research and biotechnological applications:

• Domain Engineering:

  • The successful creation of the TmTop65-EcCTD chimera demonstrates the feasibility of domain swapping to modify enzyme properties

  • Designing chimeras with domains from different thermophilic species could fine-tune temperature optima while maintaining thermostability

  • Creating libraries of C-terminal domain variants could generate enzymes with altered DNA sequence preferences

• Structure-Guided Mutagenesis:

  • Introducing additional salt bridges similar to those identified in other T. maritima thermostable proteins

  • Engineering disulfide bonds at strategic positions to enhance stability without compromising flexibility required for catalysis

  • Modifying the DNA binding interface to alter substrate specificity while maintaining catalytic efficiency

• Catalytic Enhancement:

  • Targeted mutations in the active site to increase reaction rate while maintaining thermostability

  • Modifications to enhance processivity (multiple reaction cycles per binding event)

  • Engineering allosteric sites for regulation of enzyme activity

• Application-Specific Modifications:

  • Fusion to DNA-binding domains for targeted activity at specific genomic regions

  • Introduction of reporter tags for tracking enzyme activity in complex systems

  • Development of temperature-sensitive variants for controlled activity in biotechnological processes

The chimeric topoisomerase studies described in the search results provide proof-of-concept that such engineering approaches can successfully modify the properties of T. maritima topoisomerase I, particularly cleavage specificity and DNA binding efficiency . The inherent thermostability of this enzyme makes it an excellent scaffold for engineering novel functionality while maintaining robust performance under demanding conditions.