Recombinant Neurospora crassa DNA repair protein rad-5 (rad-5), partial

What is the Neurospora crassa rad-5 protein and what are its alternative designations?

Neurospora crassa rad-5 is a DNA repair protein that is also known as mus-41. It represents the functional homolog of the RAD5 protein found in Saccharomyces cerevisiae and other fungi. The protein is characterized as a key component in the postreplication repair (PRR) pathway and is identified by gene names rad-5, mus-41, or rad5 in research literature .

How does rad-5/mus-41 fit into DNA repair pathways in Neurospora crassa?

The rad-5/mus-41 protein functions primarily in the error-free branch of postreplication repair in Neurospora crassa. Genetic analyses have demonstrated that it works downstream of uvs-2 (the RAD18 homolog), as uvs-2 is epistatic to mus-41. This positioning suggests that rad-5/mus-41 is activated after the initial recognition of DNA damage by the uvs-2 protein. Furthermore, rad-5/mus-41 operates independently from the translesion synthesis pathway mediated by upr-1 (REV3 homolog), confirming its specific role in the error-free bypass mechanism .

What types of DNA damage can rad-5/mus-41 respond to?

Research has shown that rad-5/mus-41 mutants display sensitivity to multiple DNA-damaging agents, particularly ultraviolet (UV) radiation and methyl methanesulfonate (MMS). This sensitivity profile indicates that rad-5/mus-41 plays a critical role in repairing UV-induced photoproducts (primarily cyclobutane pyrimidine dimers and 6-4 photoproducts) and alkylation damage caused by MMS .

What are the optimal expression systems for producing recombinant Neurospora crassa rad-5 protein?

Recombinant Neurospora crassa rad-5 protein can be expressed in multiple host systems including E. coli, yeast, baculovirus, and mammalian cell systems. When selecting an expression system, researchers should consider the following factors:

• E. coli systems: Provide high protein yields but may lack post-translational modifications

• Yeast systems: Better for maintaining fungal protein folding patterns

• Baculovirus systems: Superior for large proteins with complex domains

• Mammalian systems: Optimal for preserving authentic eukaryotic modifications

For functional studies, expression systems that preserve the native conformation and enzymatic activity should be prioritized. Standard purification protocols can achieve ≥85% purity as determined by SDS-PAGE .

How can researchers effectively generate and characterize rad-5/mus-41 mutants?

To generate rad-5/mus-41 mutants for functional characterization studies, researchers should employ the following methodological approach:

• Design targeted mutations in key domains (similar to the ATPase or RING domain mutations characterized in S. cerevisiae Rad5)

• Utilize CRISPR-Cas9 or traditional homologous recombination techniques for precise genetic modifications

• Verify mutations through sequencing and expression analysis

• Assess functionality through DNA damage sensitivity assays using:

  • UV radiation (254 nm, 10-100 J/m²)

  • MMS treatment (0.01-0.1%)

  • Other DNA-damaging agents for comprehensive characterization

Phenotypic characterization should include growth inhibition measurements, survival rate calculations, and microscopic examination of cellular morphology following DNA damage .

What analytical methods are most effective for studying rad-5/mus-41 protein interactions?

For investigating protein-protein and protein-DNA interactions involving rad-5/mus-41, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): To identify stable protein interaction partners

• Yeast two-hybrid or split-ubiquitin assays: For detecting direct protein interactions

• Chromatin immunoprecipitation (ChIP): To analyze rad-5/mus-41 association with DNA

• Biolayer interferometry or surface plasmon resonance: For quantitative kinetic binding analysis

• Mass spectrometry following affinity purification: To identify the complete interactome

When designing interaction studies, consider the potential involvement of rad-5/mus-41 in complexes with other DNA repair proteins, particularly those in the error-free postreplication repair pathway and potential ubiquitylation machinery components .

What is the role of rad-5/mus-41 in PCNA ubiquitylation and how does it differ from its homologs in other species?

Unlike its Saccharomyces cerevisiae homolog, Neurospora crassa rad-5/mus-41 is not essential for the ubiquitylation of Proliferating Cell Nuclear Antigen (PCNA) in response to UV damage. This significant functional difference suggests the existence of another ubiquitin ligase in N. crassa that can catalyze PCNA ubiquitylation. This functional divergence represents an important species-specific adaptation in the DNA damage response pathway.

The methodological approach to investigate this phenomenon should include:

• In vivo ubiquitylation assays using epitope-tagged PCNA

• Comparative analysis between wild-type and rad-5/mus-41 mutant strains

• Western blot analysis with anti-ubiquitin antibodies following UV exposure

• Mass spectrometry to identify alternative ubiquitin ligases in rad-5/mus-41 mutants

How does the domain architecture of rad-5/mus-41 contribute to its function?

Based on homology with S. cerevisiae Rad5, the N. crassa rad-5/mus-41 protein likely contains:

• An ATPase domain with helicase activity

• A RING domain with E3 ubiquitin ligase functionality

Research in S. cerevisiae has demonstrated that mutations in either domain result in intermediate DNA damage sensitivity compared to complete deletion mutants. This suggests both domains make significant but partially independent contributions to Rad5 function. For N. crassa, targeted mutation studies of the equivalent domains should be conducted to determine if this functional separation is conserved .

The recommended experimental approach includes:

• Generation of ATPase-dead and RING-inactive point mutants

• Complementation assays in rad-5/mus-41 null backgrounds

• DNA damage sensitivity profiling across different damaging agents

• Analysis of genetic interactions with other repair pathway components

What is the relationship between rad-5/mus-41 and interstrand cross-link repair?

Research in S. cerevisiae has demonstrated that Rad5 plays a critical role in interstrand cross-link (ICL) repair pathways that involve proteins homologous to human Fanconi anemia proteins. While direct evidence for this function in N. crassa is limited, the conservation of rad-5/mus-41 suggests it may perform similar functions.

To investigate this relationship in N. crassa, researchers should:

• Test rad-5/mus-41 mutant sensitivity to ICL-inducing agents (e.g., nitrogen mustard, mitomycin C, cisplatin)

• Perform epistasis analysis with known or predicted ICL repair genes in N. crassa

• Assess double-strand break formation in rad-5/mus-41 mutants following ICL damage

• Examine the potential interaction between rad-5/mus-41 and homologs of FANCM, FANCJ, MHF1, and MHF2 if present in N. crassa

How does rad-5/mus-41 function compare to its homologs in other fungal species?

Comparative analysis between Neurospora crassa rad-5/mus-41 and its homologs in other fungi reveals both conserved and divergent functions:

Researchers should address these comparative aspects through:

• Complementation studies across species

• Domain swap experiments to identify functional determinants

• Comparative genomics to identify co-evolved partners

• Analysis of selection pressures on different domains

What insights can be gained from comparing rad-5/mus-41 to its human homologs?

While direct human homologs of rad-5/mus-41 are not well-characterized, functional parallels exist with several human DNA repair proteins:

• HLTF (Helicase-Like Transcription Factor) shares structural and functional similarities with fungal Rad5 proteins

• SHPRH (SNF2 histone linker PHD RING helicase) also functions as a Rad5 ortholog in humans

To leverage these relationships for translational insights:

• Conduct comparative structural analysis focused on ATPase and RING domains

• Examine functional complementation potential in heterologous systems

• Investigate shared protein interaction networks

• Compare damage response specificities and mechanisms

These comparisons may reveal evolutionarily conserved mechanisms while highlighting adaptations specific to human systems versus fungal systems .

How might rad-5/mus-41 be involved in managing conflicts between replication and transcription?

DNA repair proteins often play roles in resolving conflicts between replication and transcription machinery. For rad-5/mus-41, this remains an unexplored frontier. Researchers should investigate:

• Whether rad-5/mus-41 localizes to sites of replication-transcription conflicts

• If rad-5/mus-41 mutants show increased R-loop formation or transcription-associated recombination

• Potential interactions between rad-5/mus-41 and RNA processing factors

• The effect of transcription inhibitors on rad-5/mus-41-dependent repair efficiency

Methodological approaches should include:

• ChIP-seq analysis following replication stress induction

• R-loop detection using S9.6 antibody in wild-type vs. mutant strains

• Genetic interaction screens with transcription and RNA processing mutants

• DNA-RNA hybrid mapping techniques

What role might rad-5/mus-41 play in the ecological interactions between Neurospora and bacterial species?

Recent research has identified ecological associations between Neurospora species and Pseudomonas bacteria in natural environments. This raises questions about whether DNA repair systems like rad-5/mus-41 might be involved in managing DNA damage arising from microbial interactions.

To investigate this ecological dimension:

• Compare rad-5/mus-41 expression patterns during Pseudomonas interactions

• Assess whether bacterial metabolites or secretions induce DNA damage requiring rad-5/mus-41

• Evaluate rad-5/mus-41 mutant fitness in co-culture with bacteria compared to wild-type

• Investigate potential horizontal gene transfer events that might be influenced by rad-5/mus-41 activity

This ecological perspective could reveal previously unrecognized selective pressures shaping the evolution of DNA repair systems in fungi .

How does rad-5/mus-41 interact with the alternative ubiquitin ligase in N. crassa PCNA ubiquitylation?

Given that rad-5/mus-41 is not essential for PCNA ubiquitylation in N. crassa, identifying and characterizing the alternative ubiquitin ligase represents a significant research opportunity. Researchers should:

• Perform proteomic screening to identify E3 ligases that associate with PCNA after DNA damage

• Conduct systematic genetic screens for suppressors of rad-5/mus-41 mutant sensitivity

• Investigate potential redundancy mechanisms through synthetic genetic array analysis

• Examine the ubiquitylation patterns in various genetic backgrounds to determine if different ligases generate distinct ubiquitin chain topologies

This investigation could reveal novel regulatory mechanisms controlling the choice between different repair pathways and potentially identify new therapeutic targets for DNA damage-related disorders .