Recombinant Debaryomyces hansenii Putative DNA helicase INO80 (INO80), partial

What is the INO80 complex and what are its primary functions?

The INO80 complex is a 15-subunit ATP-dependent chromatin remodeler that plays essential roles in fundamental nuclear processes. It regulates gene expression, DNA repair, and replication through three primary mechanisms: sliding nucleosomes, exchanging histone H2A.Z with H2A, and positioning +1 and -1 nucleosomes at promoter DNA . The complex functions by harnessing the energy from ATP hydrolysis to remodel chromatin structure, making DNA accessible for transcription machinery, repair proteins, and replication factors.

The INO80 complex is evolutionarily conserved across eukaryotes, suggesting its fundamental importance in chromatin regulation. For researchers working with Debaryomyces hansenii INO80, understanding these core functions provides essential context for experimental design and interpretation.

What is the structural organization of the INO80 complex?

The INO80 complex exhibits a sophisticated structural organization revealed by cryo-electron microscopy studies at 4.3Å resolution (with parts at 3.7Å). When bound to a nucleosome, the complex:

• Cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts

• Contains a Rvb1/2 AAA+ ATPase hetero-hexamer that serves as an assembly scaffold and acts as a stator for the motor and nucleosome gripping subunits

• Positions its Swi2/Snf2 ATPase motor at SHL-6, where it unwraps ~15 base pairs of DNA and disrupts H2A:DNA contacts

• Utilizes Arp5-Ies6 to grip SHL-2/-3, serving as a counter grip for the motor on the opposite side of the H2A/H2B dimer

• Features an Arp5 insertion domain that forms a "grappler" element binding the nucleosome dyad

This architecture enables INO80 to effectively remodel nucleosomes through a unified mechanism for sliding and histone editing.

How does the INO80 complex remodel nucleosomes mechanistically?

The INO80 complex employs a ratchet-like mechanism for nucleosome sliding and histone editing, as revealed by structural and biochemical analyses:

• The motor (Ino80 ATPase) binds at SHL-6, unwrapping entry DNA and disrupting H2A:DNA contacts

• ATP hydrolysis drives the motor to pump DNA into the nucleosome against the Arp5-Ies6 counter grip

• DNA groove tracking creates a loop limited between the motor and Arp5-Ies6

• This persistently disrupts the H2A/H2B DNA interface, enabling histone exchange

• When sufficient force accumulates from multiple small motor steps, the pumped DNA propagates across Arp5-Ies6 and the grappler (the ratchet step)

• This results in nucleosome movement in larger steps of 10-20bp

During this process, the grappler ensures structural integrity of the octamer by holding onto H2A/H2B at the site where entry DNA unwraps, while Ies2 binds the acidic patch on the other side and acts as a throttle for the ATPase.

What role does the HSA domain play in INO80 function?

The helicase-SANT-associated (HSA) domain serves as the primary binding platform for nuclear actin-related proteins (ARPs) and actin within the INO80 complex. Research findings indicate that:

• The HSA domain of Ino80 (amino acids 462-598) is sufficient for binding Arp4, actin, and Arp8

• Each HSA domain exhibits remarkable specificity; no actin associates with the HSA from Sth1, and no Arp7 or Arp9 associates with HSA domains from Eaf1, Swr1, or Ino80

• Secondary structure predictions suggest HSA domains form long α-helices

• The HSA likely evolved to bind ARP-ARP or ARP-actin dimer units rather than selecting them individually

This domain architecture appears conserved across multiple chromatin remodeling complexes that contain ARPs, including SWR1 and INO80, suggesting a fundamental organizational principle for nuclear ARP-containing complexes.

How is histone variant exchange mediated by INO80?

The INO80 complex facilitates the exchange of histone variants, particularly replacing H2A.Z with H2A, through a sophisticated mechanism:

• The initial binding of the Ino80 ATPase at SHL-6 unwraps ~15bp of entry DNA

• This unwrapping breaks DNA contacts with H2A L2 (loop 2) at SHL-5.5 and H3 (helix αN) at SHL-6.5

• ATP-driven DNA translocation further disrupts H2A's and H2B's L1 and α1 DNA contacts, partially exposing the H2A/H2B dimer

• The grappler element maintains octamer integrity during this process by holding onto H2A/H2B

• The grappler "foot" acts as a sensor that binds H2A at a region where H2A and H2A.Z differ in amino acid sequence

Experimental evidence supports this model: introducing H2A.Z-mimicking mutations into H2A at the interface with the foot increased sliding velocity, consistent with the faster sliding of H2A.Z nucleosomes by INO80 .

What expression systems are recommended for recombinant INO80 complex production?

For researchers working with Debaryomyces hansenii INO80, the baculovirus expression system in insect cells has proven effective for recombinant INO80 production based on studies with other species:

• Clone genes encoding INO80 components into appropriate vectors:

  • pACEBac1, pIDC, and pIDK vectors for different subunits

  • Combine vectors into bacmids using Cre recombinase-mediated recombination

• Generate baculoviruses in Spodoptera frugiperda (SF21) insect cells

• Express proteins in Trichoplusia ni insect cells for optimal yield

This approach enables production of stoichiometric complexes that stably bind and remodel nucleosomes, consistent with activities observed in human and S. cerevisiae INO80 complexes.

What purification strategies yield functional INO80 complex?

Affinity chromatography approaches have successfully yielded functional INO80 complex components:

• Express tagged versions of INO80 components (10×His and Flag tags are effective)

• Perform sequential purification:

  • Ni-NTA chromatography for His-tagged proteins

  • Anti-Flag chromatography for Flag-tagged proteins

• Verify purified components by:

  • Mass spectrometry sequencing

  • Immunoblot analysis

  • Protein staining

These approaches yield near-homogeneous protein preparations suitable for structural and functional studies. When purifying from D. hansenii, researchers should consider species-specific optimization of buffer conditions and expression parameters.

How can one assess the nucleosome remodeling activity of recombinant INO80?

Multiple complementary assays can effectively measure INO80's nucleosome remodeling activities:

• Nucleosome sliding assays:

  • Using defined nucleosome substrates (e.g., assembled with Widom 601 sequence)

  • Measuring position changes via native gel electrophoresis

  • Quantifying sliding rates under different conditions

• Histone exchange assays:

  • Using differentially labeled H2A/H2B dimers

  • Monitoring exchange through FRET or other spectroscopic methods

• ATPase activity measurements:

  • Testing ATP hydrolysis with various nucleosome substrates

  • Comparing rates between wild-type and mutant INO80 complexes

  • Assessing nucleosome-stimulated versus basal ATPase activity

When characterizing D. hansenii INO80, comparing its kinetic parameters with those of well-studied orthologs provides valuable insights into functional conservation and specialization.

Which domains of the Ino80 ATPase are critical for specific functions?

The Ino80 ATPase contains several functionally specialized domains:

• Swi2/Snf2 ATPase motor:

  • N and C-lobes engage with double-stranded DNA

  • Brace helix I reaches across both lobes, stabilizing their mutual orientation

  • Functions as the primary DNA translocase that drives nucleosome remodeling

• HSA domain (in Ino80: amino acids 462-598):

  • Primary binding platform for Arp4, Arp8, and actin

  • Essential for complex assembly and stability

• Insertion domain (~270 amino acid segment in C-lobe):

  • Adopts a wheel-like structure

  • Sequentially binds to all six Rvb1/2 protomers in the central cavity

  • Contains a "latch" that generates a distinct interaction site for Arp5-Ies6

  • Stimulates Rvb1/2's ATP hydrolysis activity 16-fold

Understanding these domain functions is crucial for designing targeted mutations to probe specific aspects of INO80 function in D. hansenii.

How do mutations in key components affect INO80 activity?

Mutations in specific regions of the INO80 complex have revealed important structure-function relationships:

• Acidic patch targeting mutations:

  • Mutations affecting both Ies2 and Arp5 grappler interactions with the nucleosome acidic patch

  • Abrogated nucleosome sliding while only moderately reducing ATPase rates

  • Demonstrates the critical role of these interactions in coupling ATP hydrolysis to effective remodeling

• H2A mutations at the grappler foot interface:

  • H2A.Z-mimicking mutations increased sliding velocity

  • Suggests this interface serves as a sensor for histone variant identity

  • Explains faster sliding of H2A.Z nucleosomes by INO80

• HSA domain mutations:

  • Alter ARP binding specificity

  • Affect complex assembly and stability

These findings provide a framework for targeted mutagenesis in D. hansenii INO80 to probe conservation of these functional relationships.

How might D. hansenii INO80 differ from well-characterized orthologs?

While specific data on D. hansenii INO80 is limited in the provided search results, comparative analysis suggests potential differences:

• Subunit composition:

  • Core catalytic components (Ino80 ATPase, Rvb1/2, ARPs) are likely conserved

  • Species-specific accessory subunits may exist (comparable to Ies1, 3, 5, Nhp10 in S. cerevisiae)

  • These differences could confer specialized regulatory properties

• Sequence variations in key interfaces:

  • HSA domain variations may affect ARP binding specificity

  • Grappler domain variations could alter histone variant discrimination

  • ATPase domain differences might modify catalytic parameters

• Post-translational modifications:

  • Species-specific regulatory modifications might exist

  • Could adapt INO80 function to D. hansenii's environmental niche

Researchers should consider these potential differences when designing experiments and interpreting results with D. hansenii INO80.

What evolutionary insights can be gained from studying D. hansenii INO80?

Studying D. hansenii INO80 in comparison to other species offers valuable evolutionary insights:

• Conservation of core mechanisms:

  • The ratchet-like mechanism of nucleosome sliding appears fundamental across species

  • The HSA-ARP-actin module organization is evolutionarily conserved

• Adaptation to genomic context:

  • D. hansenii's adaptations to high-salt environments may be reflected in INO80 properties

  • Could reveal how chromatin remodeling adapts to different genomic GC content and organization

• Lineage-specific innovations:

  • Comparing D. hansenii INO80 with other yeast species could highlight lineage-specific features

  • May reveal how chromatin remodeling contributes to speciation and adaptation

This comparative approach provides context for understanding fundamental versus specialized aspects of INO80 function across eukaryotes.

How can structural information guide design of INO80 inhibitors?

The detailed structural information on INO80-nucleosome interactions provides a foundation for rational inhibitor design:

• Target sites for inhibition:

  • The ATPase domain at SHL-6 - small molecules could block ATP binding or hydrolysis

  • The Arp5-Ies6 interaction with SHL-2/-3 - peptides or compounds could disrupt this counter grip

  • The grappler-nucleosome dyad interface - molecules targeting this interaction could prevent proper engagement

• Potential specificity determinants:

  • The unique conformation of DNA at the motor binding site (widened minor groove)

  • The specific contacts between the grappler foot and H2A/H2B

  • The HSA domain-ARP interfaces

Developing such inhibitors would provide valuable tools for dissecting INO80 functions in D. hansenii and potentially therapeutic approaches for fungal infections.

What techniques enable real-time monitoring of INO80 activity?

Advanced biophysical techniques enable real-time monitoring of INO80 remodeling activities:

• Single-molecule approaches:

  • Optical tweezers to measure force generation during remodeling

  • FRET-based assays to track conformational changes in DNA and histones

  • DNA curtain assays to visualize multiple remodeling events simultaneously

• Advanced microscopy:

  • Super-resolution imaging of INO80-chromatin interactions

  • Live-cell tracking of labeled INO80 during DNA repair or replication

• Specialized biochemical assays:

  • Real-time monitoring of ATP hydrolysis coupled to nucleosome movement

  • Stopped-flow kinetics to measure rapid conformational changes

  • Chemical crosslinking coupled with mass spectrometry to capture transient interactions

These approaches would provide unprecedented insights into the dynamics of D. hansenii INO80 activity.