Recombinant Sulfolobus acidocaldarius DNA double-strand break repair Rad50 ATPase (rad50), partial

What is the function of Rad50 ATPase in Sulfolobus acidocaldarius?

Rad50 ATPase in S. acidocaldarius is a component of the Rad50/Mre11 complex that plays a critical role in the initial stages of DNA double-strand break (DSB) repair. The complex is responsible for DNA end recognition, binding, and processing to facilitate subsequent repair. Rad50's coiled-coil regions transiently bind DNA, enabling the complex to traverse DNA until encountering a free end (DSB). Following detection, Rad50 facilitates the recruitment of additional repair proteins including HerA and NurA, which resect DNA ends to generate single-stranded regions necessary for homologous recombination. The Rad50-Mre11 complex also bridges DNA ends, preventing chromosomal fragmentation during the repair process.

How are double-strand breaks formed in Sulfolobus species?

In Sulfolobus species, DSBs can arise through multiple mechanisms:

• UV irradiation - Studies with S. solfataricus have shown that UV-C exposure results in the formation of DSBs, likely arising from unrepaired lesions (such as cyclobutane pyrimidine dimers and 6-4 pyrimidone photoproducts) that remain as cells progress through replication .

• Environmental stressors - The natural hot spring habitat of Sulfolobus (70-85°C) leads to DNA damage through generation of reactive oxygen species and hydrolytic deamination of nucleotide bases .

• Replication stress - When DNA lesions remain unrepaired as cells progress through DNA replication, this can lead to replication fork collapse and subsequent DSB formation .

DSBs can be directly visualized using pulsed-field gel electrophoresis (PFGE), which has demonstrated repair rate variations among different Sulfolobus strains following UV-C irradiation .

What are the key functional domains of recombinant Rad50?

The recombinant Sulfolobus acidocaldarius Rad50 protein contains several functional domains essential for its activity:

The protein exhibits ATP-dependent DNA unwinding capabilities, extending single-stranded regions by hundreds of base pairs. It interacts with multiple partner proteins (Mre11, HerA, NurA) to coordinate the DSB repair process, and the native protein maintains thermal stability at temperatures as high as 60°C.

How does the ATPase activity of Rad50 regulate DNA repair mechanisms?

The ATPase activity of Rad50 plays a critical regulatory role in DNA repair through a sophisticated ATP-binding and hydrolysis cycle:

This regulated cycle of ATP binding and hydrolysis effectively coordinates the sequential steps of DSB processing and repair.

What is the significance of having two ATPase sites in the Rad50 complex?

Research using catalytic site mutants has demonstrated that both ATPase sites in the Rad50 dimer are critical for proper function:

• Symmetrical engagement: Studies show that symmetrical engagement of both Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells .

• Concerted hydrolysis: Experiments with human MRN complexes containing only one functional ATPase site revealed that concerted hydrolysis at both sites is essential for:

  • DNA-stimulated ATP hydrolysis

  • Endonucleolytic cleavage of DNA at protein adducts

  • ATM kinase activation

• Conformational changes: The binding of ATP at both sites promotes the "closed" conformation necessary for DNA binding and tethering, while hydrolysis at both sites is required for the conformational changes that enable Mre11 nuclease activity .

This requirement for dual ATPase functionality suggests an evolutionarily conserved mechanism for regulating the molecular machine that processes DNA breaks, ensuring precise control over the repair process.

How does Rad50 function in the UV damage response of Sulfolobus acidocaldarius?

In S. acidocaldarius, Rad50 plays a specialized role in the response to UV damage:

• Community-based repair: Following UV exposure, S. acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair DSBs via homologous recombination .

• Association with damaged DNA: The Mre11 protein from S. acidocaldarius has been found to associate with DNA following damage and directly interacts with Rad50, indicating a role for both proteins in break repair .

• Facilitating genetic exchange: UV treatment of S. acidocaldarius mutants increases the rate of exchange of genetic markers, suggesting that DNA lesions and DSBs stimulate this process .

• Coordinated response: UV damage in Sulfolobus results in the upregulation of several genes involved in DSB repair, with distinct temporal expression patterns across different strains .

The Rad50-Mre11 complex appears to be central to this unique UV response mechanism that combines cellular aggregation, DNA exchange, and homologous recombination to maintain genomic integrity under extreme environmental conditions.

How does Rad50's function compare between archaea and eukaryotes?

Rad50's fundamental mechanisms show both conservation and divergence between archaeal and eukaryotic systems:

The Mre11-Rad50 core complex is so fundamental to repair that it is conserved across bacteriophages, bacteria, archaea, and eukaryotes , underscoring its evolutionary importance in genome maintenance.

What techniques are used to assess Rad50's ATPase activity?

Several complementary techniques are employed to assess the ATPase activity of Rad50:

• Phosphate release assays:

  • Measurement of inorganic phosphate (Pi) release to quantify ATP hydrolysis rates

  • Can be used to assess the effects of DNA, protein partners, and mutations on hydrolysis rates

• ATP binding assays:

  • Incubation of purified Rad50 with [γ-32P]ATP followed by UV irradiation

  • SDS-PAGE analysis to visualize ATP binding through autoradiography

  • Quantitative analysis to determine concentration-dependent binding

• Thin-layer chromatography:

  • Used to explore ATP hydrolysis catalyzed by Rad50

  • Can reveal the effects of interacting proteins on hydrolysis rates

• ATP hydrolysis kinetics:

  • Studies with varying DNA concentrations show stimulation patterns

  • For example, addition of 0.5 nM DNA to P. furiosus MR complex resulted in a 12-14 fold increase in ATP hydrolysis

These techniques have revealed that double-stranded DNA stimulates ATP hydrolysis in an end-dependent manner and that both ATPase sites in the Rad50 dimer are required for optimal activity.

How can researchers visualize and quantify DNA double-strand breaks and their repair in Sulfolobus species?

Researchers employ several techniques to visualize and quantify DSBs and their repair in Sulfolobus:

• Pulsed-field gel electrophoresis (PFGE):

  • Directly visualizes the state of chromosomes following damage

  • Can detect fragmentation and subsequent repair

  • Has demonstrated repair rate variations among different Sulfolobus strains following UV-C irradiation

  • CHEF (Contour-clamped homogeneous electric field) and TAFE (Transverse alternating-field electrophoresis) are specialized PFGE methods that provide different resolution capabilities

• Mung Bean Nuclease (MBN) treatment:

  • Treatment of DNA samples with MBN before electrophoresis

  • Can distinguish between resected and unresected DNA breaks

  • Decrease in band intensity after MBN treatment indicates resection has occurred

• Survival curves:

  • Quantification of cellular recovery after UV exposure

  • Can reveal strain-specific differences in DNA repair efficiency

• Transcriptional analysis:

  • Examination of gene expression changes following DNA damage

  • Has identified 55 UV-responsive genes in S. solfataricus with pronounced changes in mRNA copy numbers over extended periods

These methods collectively provide insights into both the physical state of damaged DNA and the cellular responses to DNA damage in these extremophilic organisms.

What methods are used to study the interaction between Rad50 and other DNA repair proteins?

Researchers employ multiple complementary approaches to investigate the interactions between Rad50 and other DNA repair proteins:

• Biochemical interaction assays:

  • Purified protein binding studies to detect direct interactions

  • Have demonstrated that Rad50 facilitates the recruitment of HerA and NurA following DSB detection

  • Have shown Mre11's direct interaction with Rad50 in S. acidocaldarius following DNA damage

• Structural studies:

  • X-ray crystallography and cryo-EM to elucidate Rad50's interaction with DNA and other proteins

  • Have revealed conformational changes during ATP hydrolysis and identified the zinc-hook apex as a critical interface for DNA binding

• Genetic approaches:

  • Creation of null mutants (e.g., rad50 and mre11 nulls)

  • Functional complementation studies

  • Have shown that mre11 nulls exhibit similar magnitude of defect to rad50 nulls, supporting their function in a complex

• Real-time imaging:

  • Fast-scanning atomic force microscopy (FS-AFM)

  • Enables visualization of Rad50's DNA binding and translocation in real time

• ATP-dependent functional assays:

  • Assessment of how ATP binding and hydrolysis affect protein-protein interactions

  • Have demonstrated that ATP binding creates a "closed" conformation necessary for certain protein interactions

These methods have collectively revealed the dynamic nature of Rad50's interactions with repair machinery and how these interactions are regulated by ATP binding and hydrolysis.

How can researchers generate and characterize rad50 null mutants to study its function?

Generation and characterization of rad50 null mutants involves several key methodological steps:

• Gene deletion approach:

  • Sequential replacement of gene alleles with antibiotic resistance cassettes

  • For example, replacement with NEO and BLA resistance markers

  • Verification through PCR analysis to confirm rad50 loss and replacement

• Growth and survival assessment:

  • Monitoring growth curves with and without DNA damage induction

  • Quantification of cloning efficiency (percentage of cells surviving cloning)

  • Measurement of normalized survival efficiency following induced DSBs

• DSB induction methods:

  • Use of I-SceI meganuclease for targeted DSB creation

  • γ-radiation for random DSB induction

  • UV-C exposure for damage that can lead to DSB formation

• Characterization of repair defects:

  • Assessment of DSB resection dynamics (often surprisingly altered in nulls)

  • Evaluation of RAD51 focus formation (typically compromised in rad50 nulls)

  • Analysis of repair pathway choice (shift from HR to MMEJ in rad50 nulls)

• Functional comparisons:

  • Parallel generation of related mutants (e.g., mre11 nulls) for comparison

  • Assessment of epistatic relationships with other repair genes

These approaches have revealed that rad50 null mutants typically show severe growth defects, impaired DSB repair, compromised DNA damage response signaling, and altered repair pathway choice, collectively highlighting Rad50's critical role in DNA repair mechanisms.

How can recombinant Rad50 ATPase be utilized in biotechnology applications?

Recombinant Rad50 ATPase from S. acidocaldarius offers several promising biotechnology applications:

• Thermostable enzyme technology:

  • The native protein's stability at 60°C makes it valuable for high-temperature applications

  • Potential use in PCR and other amplification technologies requiring thermostable components

• Genome editing enhancement:

  • Potential to improve efficiency of genome editing tools by facilitating DNA repair processes

  • Could enhance homologous recombination-based editing approaches

• DNA manipulation and analysis:

  • ATP-dependent DNA unwinding capability extends single-stranded regions by hundreds of base pairs

  • Potential applications in DNA preparation for sequencing and other analytical methods

• Model system for drug discovery:

  • Provides a platform for screening compounds that target DNA repair proteins

  • Could lead to development of novel antimicrobials targeting archaeal-specific repair mechanisms

• Understanding extremophile adaptation:

  • Studies with recombinant Rad50 can provide insights into molecular mechanisms of adaptation to extreme environments

  • May inspire biomimetic approaches to creating stress-resistant systems

Future research may further explore these applications, potentially expanding the toolkit available for molecular biology and biotechnology.

What are the outstanding questions regarding Rad50's role in archaeal DNA repair?

Despite significant advances, several key questions about Rad50's function in archaeal DNA repair remain:

• Regulatory mechanisms:

  • How is Rad50's ATPase activity precisely regulated in the cellular context?

  • Are there archaeal-specific factors that modulate its activity?

  • What triggers ATP hydrolysis in vivo?

• Evolutionary considerations:

  • How have archaeal Rad50 proteins evolved different properties (e.g., higher ATPase rates) compared to their eukaryotic counterparts?

  • What selective pressures in extreme environments have shaped Rad50 function?

• System-specific processes:

  • How does Rad50 contribute to the community-based DNA repair mechanism in Sulfolobus?

  • What is the precise sequence of molecular events during cell aggregation and DNA exchange following UV damage?

• Structural dynamics:

  • What are the detailed conformational changes that occur during the ATP binding and hydrolysis cycle?

  • How do these changes coordinate the sequential steps of DSB processing?

• Integration with other repair pathways:

  • How does Rad50-mediated repair interact with other DNA repair mechanisms in archaea?

  • Are there archaeal-specific backup pathways when Rad50 function is compromised?

Addressing these questions will require combining structural biology, biochemistry, genetics, and systems approaches to develop a comprehensive understanding of archaeal DNA repair mechanisms.

How do different Sulfolobus strains vary in their DNA repair capabilities?

Research has revealed significant strain-specific variations in DNA repair capabilities among Sulfolobus species:

• Survival and recovery differences:

  • S. solfataricus strain P2 obtained from different sources and strain 98/2 show different survival curves and recovery abilities following UV-C irradiation

  • Recovery capacity depends on both strain type and radiation dose applied

• Chromosomal repair rate variations:

  • Direct visualization using pulsed-field gel electrophoresis demonstrates different repair rates following UV-C-induced DSBs among strains

  • The timing and efficiency of DSB repair varies significantly between strains

• Transcriptional response differences:

  • Several genes involved in DSB repair show significant upregulation after UV-C irradiation

  • Transcript abundance levels and temporal expression patterns for DSB repair genes are distinct for each strain

  • This indicates strain-specific regulatory networks governing the DNA damage response

• Repair pathway preferences:

  • At low UV doses, homologous recombination appears to be the preferred repair mechanism

  • At higher doses, photoreactivation becomes a major mechanism in some strains

  • The balance between these pathways differs between strains

These variations suggest that even closely related Sulfolobus strains have evolved distinct DNA repair strategies, possibly reflecting adaptation to slightly different environmental niches or evolutionary histories.

What insights can archaeal Rad50 provide for understanding eukaryotic DNA repair mechanisms?

Studying archaeal Rad50 offers valuable insights into eukaryotic DNA repair through several comparative angles:

• Conserved core mechanisms:

  • The Mre11-Rad50 core complex is conserved across all domains of life, suggesting fundamental mechanistic principles

  • Studies in archaea can reveal these core functions without the complexity of additional eukaryotic components

• ATP regulation similarities:

  • Both archaeal and eukaryotic Rad50 show DNA-stimulated ATP hydrolysis

  • Both require dual ATPase functionality for optimal activity

  • These similarities suggest conserved regulatory principles

• Evolutionary insights:

  • Archaea are evolutionarily closer to eukaryotes than bacteria

  • Comparing archaeal and eukaryotic Rad50 function can reveal how DNA repair mechanisms evolved with increasing cellular complexity

• Structural simplicity advantage:

  • Archaeal complexes often lack some regulatory components present in eukaryotes

  • This simplicity facilitates structural studies that would be challenging with eukaryotic complexes

  • Structures of archaeal complexes can serve as templates for understanding eukaryotic counterparts

• Functional adaptations:

  • Different rates of ATP hydrolysis between archaeal and eukaryotic Rad50 may reflect adaptation to different cellular environments

  • Understanding these adaptations can reveal principles of enzyme evolution and optimization