Recombinant Saccharomyces cerevisiae Flap endonuclease 1 (RAD27)

What is the structural and functional relationship between yeast RAD27 and human FEN1?

RAD27 (also called RTH1) is the Saccharomyces cerevisiae homolog of human FEN1 (Flap Endonuclease 1), displaying remarkable conservation with 79% conserved and 60% identical amino acids . This high degree of conservation underscores the fundamental importance of this nuclease across eukaryotes. Both proteins function as structure-specific 5'-3' flap exo/endonucleases that recognize and cleave specialized DNA structures that arise during DNA replication and repair processes .

The primary functions of RAD27/FEN1 include processing the 5' ends of Okazaki fragments during DNA replication, participating in base excision repair, and maintaining genomic stability . This functional conservation makes yeast RAD27 an excellent model for understanding human FEN1 activity, with insights potentially applicable to human disease contexts, particularly those involving trinucleotide repeat expansions associated with neurodegenerative disorders .

What phenotypes are associated with RAD27 deletion in yeast?

Deletion of RAD27 (rad27Δ) in S. cerevisiae results in multiple distinct phenotypes that highlight its crucial roles in DNA metabolism:

These phenotypes reflect RAD27's diverse roles in maintaining genome integrity through multiple mechanisms . Importantly, the conditional lethality of rad27Δ mutants indicates that other nucleases or alternative pathways can partially compensate for RAD27's absence under permissive conditions .

How do the rad27-G67S and rad27-G240D mutations affect enzyme function?

The rad27-G67S and rad27-G240D mutations provide valuable insights into RAD27's structure-function relationships:

Both mutations confer defective substrate cleavage and a dinucleotide repeat expansion mutator phenotype, but with distinct characteristics . The rad27-G67S mutation appears to more severely affect repeat tract stability relative to other mutagenic events when compared to rad27-G240D . This differential effect suggests these residues contribute uniquely to processing various DNA structures.

Mechanistic studies have revealed that these mutations affect the enzyme's ability to perform "flap equilibration" rather than simply reducing general cleavage efficiency . Flap equilibration refers to the ability of a flap to partially or completely reanneal to the template, forming various double-flap intermediates with an adjacent primer . Wild-type RAD27 effectively captures cleavable flap structures among these equilibrating intermediates, while the mutants are deficient in this capacity.

This finding highlights that the critical factor in preventing trinucleotide repeat expansion is not merely cleavage efficiency, but rather the ability to capture cleavable flap structures during the dynamic equilibration process . This mechanistic insight has important implications for understanding how RAD27/FEN1 prevents genomic instability in contexts beyond simple flap processing.

What experimental approaches can distinguish between different RAD27 nuclease activities?

Several sophisticated biochemical assays can differentiate between RAD27's distinct nuclease activities:

Flap Endonuclease Activity:

• Use synthetic DNA substrates with a 5' single-stranded flap

• Incorporate fluorescent or radioactive labels at strategic positions

• Monitor cleavage at the base of the flap by gel electrophoresis

• Quantify the efficiency and specificity of cleavage

Exonuclease Activity:

• Employ nicked duplex DNA substrates

• Label the 5' end at the nick position

• Track progressive degradation from the nick

• Analyze the pattern of degradation products

R-loop Processing:

• Create RNA-DNA hybrid structures with displaced DNA strands

• Incorporate TERRA sequences to mimic telomeric R-loops

• Map cleavage sites on the RNA component

• Compare with RNase H activity for mechanistic insights

Double-flap Processing:

• Design substrates with both 5' and 3' flaps at a nick

• Vary flap lengths and compositions to test substrate preferences

• Determine how efficiently different flap configurations are processed

For all these assays, comparing wild-type RAD27 with specific mutants (e.g., rad27-G67S, rad27-G240D) can reveal how different mutations selectively affect each activity, providing insights into the structural determinants of substrate specificity .

How does RAD27 prevent trinucleotide repeat (TNR) expansion?

RAD27 prevents trinucleotide repeat expansion through a sophisticated mechanism involving flap equilibration:

During DNA replication or repair, trinucleotide repeat sequences can form unusual secondary structures in flap intermediates . These structures, if unresolved, can lead to repeat expansion upon ligation. RAD27 counters this process through several coordinated activities:

• Recognition of flap structures containing TNR sequences

• Engagement with the flap through its flap-tracking mechanism

• Facilitation of flap equilibration, allowing the flap to sample different conformations

• Capture of cleavable flap structures among the equilibrating intermediates

• Efficient endonucleolytic cleavage that prevents the formation of expansion intermediates

Research with the rad27-G67S and rad27-G240D mutants revealed that the critical factor in preventing TNR expansion is not simply cleavage efficiency, but rather the ability to capture a cleavable flap structure during equilibration . The wild-type RAD27 nuclease effectively interacts with these equilibrating TNR intermediates, leading to efficient cleavage and prevention of expansion.

This mechanism explains why RAD27/FEN1 deficiency is associated with trinucleotide repeat instability and provides insights into how similar processes might contribute to human diseases characterized by repeat expansions .

What is the relationship between RAD27 and homologous recombination pathways?

RAD27 and homologous recombination (HR) pathways share a complex relationship characterized by synthetic lethality and functional compensation:

All genes in the Rad52 recombinational repair pathway are required for the survival of rad27Δ strains at both permissive (23°C) and semipermissive (30°C) temperatures . This synthetic lethality indicates that homologous recombination serves as a critical backup pathway when RAD27 is absent. Additional mutations that confer synthetic lethality with rad27Δ include rad50S, mre11s, com1/sae2, and srs2, further emphasizing connections to DNA damage processing and recombination .

The mechanistic basis for this relationship appears to be that:

• RAD27 deficiency leads to accumulation of unprocessed Okazaki fragments and flap structures

• These structures can be converted to double-strand breaks if left unresolved

• Homologous recombination becomes essential to repair these breaks and maintain viability

• When both pathways are compromised, the accumulated DNA damage becomes lethal

This relationship reveals that homologous recombination is the primary, but not only, pathway that functions to bypass the replication defects arising in RAD27's absence . This finding has important implications for understanding synthetic lethal interactions that could be exploited in contexts where either pathway is compromised, such as in cancer cells with defects in homologous recombination.

What is the mechanism by which RAD27 regulates telomere recombination?

RAD27 plays a critical role in telomere maintenance by processing TERRA-associated R-loops:

TERRA (TElomeric Repeat-containing RNA) can form R-loops at telomeres by hybridizing with the telomeric DNA template . These R-loop structures can promote telomere recombination, particularly in telomerase-deficient cells. RAD27 functions as a negative regulator of this process through the following mechanism:

• RAD27 recognizes TERRA-associated R-loop structures at telomeres

• It selectively cleaves the RNA component (TERRA) within these structures

• This cleavage activity suppresses the accumulation of TERRA-associated R-loops

• By reducing R-loops, RAD27 negatively regulates the formation of C-circles (single-stranded C-rich telomeric DNA circles)

• C-circles are closely associated with alternative lengthening of telomeres, a recombination-based mechanism

Experimental evidence supports this model, as mutation in RAD27 results in early formation of type II recombination, confirming RAD27's role as a negative regulator in telomere recombination . In vitro assays demonstrate that RAD27 can selectively cleave TERRA in both R-loop and double-flapped structures .

This mechanism reveals how RAD27 maintains chromosome stability by restricting R-loop accumulation within the genome, particularly at telomeres, thereby preventing inappropriate recombination events that could lead to genomic instability .

How does RAD27's role at telomeres differ from its replication and repair functions?

RAD27's telomeric functions represent a specialized application of its nuclease activity that differs from its replication and repair roles in several key aspects:

Substrate Specificity:

• Telomeres: Processes RNA-DNA hybrids (R-loops) containing TERRA RNA

• Replication: Primarily cleaves DNA flaps during Okazaki fragment maturation

• Repair: Processes various DNA intermediate structures during base excision repair

Consequence of Deficiency:

• Telomeres: Accelerated telomere recombination and early formation of type II survivors

• Replication: Accumulation of unprocessed Okazaki fragments leading to replication stress

• Repair: Increased sensitivity to DNA-damaging agents and elevated mutation rates

Pathway Relationships:

• Telomeres: Acts as a negative regulator of alternative lengthening of telomeres

• Replication: Functions in the primary pathway for Okazaki fragment processing

• Repair: Participates in the long-patch branch of base excision repair

These distinctions highlight RAD27's versatility in maintaining genome integrity through multiple mechanisms . The enzyme appears to have evolved to recognize structurally similar substrates (flaps or junction points between nucleic acids) across diverse cellular contexts, allowing it to coordinate replication, repair, and telomere maintenance through related but distinct activities.

What are the optimal conditions for expressing and purifying recombinant RAD27?

Optimizing expression and purification of recombinant RAD27 requires careful consideration of several factors:

Expression Systems:

• E. coli Expression:

  • Recommended strains: BL21(DE3), Rosetta(DE3) for rare codon optimization

  • Vectors: pET series with 6xHis or GST tag

  • Induction: 0.1-0.5 mM IPTG at 16-18°C for 16-20 hours (low temperature minimizes inclusion body formation)

• Yeast Expression:

  • Using S. cerevisiae as the native host can provide proper folding and post-translational modifications

  • Consider GAL1 promoter-based vectors for inducible expression

Purification Strategy:

• Initial Capture:

  • Affinity chromatography using Ni-NTA for His-tagged RAD27

  • Consider tag removal using specific proteases if the tag affects activity

• Secondary Purification:

  • Ion-exchange chromatography (particularly Heparin columns which work well for DNA-binding proteins)

  • Size-exclusion chromatography for final polishing and to verify monodispersity

Buffer Optimization:

Quality Control:

• Assess purity by SDS-PAGE (aim for >90% purity)

• Confirm identity by Western blotting or mass spectrometry

• Verify activity using standard flap substrate cleavage assays

• Check for nuclease contamination using control substrates

These conditions should be optimized for each specific construct and application to ensure the production of properly folded, active RAD27 suitable for biochemical and structural studies.

How can researchers design physiologically relevant substrates for studying RAD27 activity?

Designing physiologically relevant substrates is crucial for accurately characterizing RAD27 activity:

Okazaki Fragment Processing Substrates:

• Standard 5' Flap Substrate:

  • Template strand: 30-50 nucleotides

  • Upstream primer: 15-25 nucleotides with 5' flap of 10-30 nucleotides

  • Downstream primer: 15-25 nucleotides annealed to template

  • Include RNA at the 5' end of the flap to mimic authentic Okazaki fragments

• Double-Flap Substrate:

  • Similar to standard flap but with 1-nucleotide 3' flap on the upstream primer

  • More closely resembles physiological intermediates during strand displacement synthesis

Trinucleotide Repeat Substrates:

• TNR Flap Structures:

  • Incorporate CAG/CTG, CGG/CCG, or GAA/TTC repeats in the flap

  • Vary repeat length (5-30 repeats) to study length-dependent effects

  • Design substrates that can form secondary structures (hairpins, loops)

R-loop Substrates:

• TERRA-containing R-loops:

  • RNA strand with telomeric repeats (UUAGGG)n

  • Complementary DNA template

  • Displaced DNA strand

  • Various lengths to test size-dependent processing

Detection Strategies:

• Fluorescent labels (FAM, Cy3, Cy5) for real-time monitoring and FRET-based assays

• Radiolabeling (32P) for highest sensitivity in gel-based assays

• Biotin-streptavidin modifications for surface immobilization in single-molecule studies

These substrate designs allow for detailed biochemical characterization of RAD27's activity on structures resembling its physiological targets . Comparing wild-type and mutant RAD27 processing of these substrates can provide mechanistic insights into how specific mutations affect different aspects of RAD27 function.

How do synthetic lethal interactions inform our understanding of RAD27 functions?

Synthetic lethal interactions provide crucial insights into RAD27's cellular roles and pathway relationships:

Key Synthetic Lethal Interactions with rad27Δ:

• Homologous recombination genes: All components of the Rad52 pathway

• DNA damage sensing/processing genes: rad50S, mre11s, com1/sae2, srs2

• Synergistic effect with rad59Δ

These interactions reveal several important aspects of RAD27 function:

• Backup Pathway Identification: The synthetic lethality with HR genes indicates that homologous recombination serves as the primary backup pathway for resolving DNA damage that accumulates in rad27Δ cells . This explains why rad27Δ cells are viable under normal conditions despite RAD27's important functions.

• Damage Type Characterization: The specific pattern of interactions helps identify what types of DNA lesions accumulate when RAD27 is missing. These likely include unprocessed Okazaki fragments, unresolved flap structures, and DNA breaks arising from replication fork collapse .

• Pathway Hierarchy: The synthetic interactions reveal which pathways operate in parallel versus serially with RAD27-dependent processes. For example, the relationship with the Rad52 pathway indicates parallel functioning, where either pathway can address certain DNA lesions .

• Functional Redundancy: The set of genes showing synthetic lethality helps identify proteins with potentially overlapping functions that can compensate for RAD27 loss under specific conditions.

This network of genetic interactions confirms that homologous recombination is the primary, but not only, pathway that functions to bypass replication defects arising in the absence of RAD27 . This understanding has important implications for predicting genetic vulnerabilities in contexts where either pathway is compromised.

What genetic assays are most informative for studying RAD27 function in vivo?

Several specialized genetic assays provide valuable insights into RAD27's diverse functions:

Mutation Rate and Spectrum Analysis:

• CAN1 Forward Mutation Assay:

  • The CAN1 gene encodes arginine permease; mutations confer canavanine resistance

  • rad27Δ mutants show characteristic duplication mutations flanked by short direct repeats

  • This assay reveals the types of genomic rearrangements that occur in RAD27's absence

• hom3-10 Frameshift Reversion Assay:

  • Specifically detects frameshift mutations in mononucleotide repeat sequences

  • Highly sensitive for detecting mismatch repair defects

  • rad27Δ shows modest increase in reversion rate, suggesting a potential role in MMR

Repeat Stability Assays:

• Dinucleotide Repeat Instability:

  • rad27Δ primarily increases insertions in dinucleotide repeats, while mismatch repair defects primarily increase deletions

  • This differential effect helps distinguish RAD27's role from classical MMR functions

• Trinucleotide Repeat Expansion:

  • Monitor stability of CAG/CTG or other trinucleotide repeats

  • rad27Δ shows increased expansion rates, reflecting its role in preventing TNR instability

Telomere Recombination Assays:

• Type II Survivor Formation:

  • In telomerase-deficient cells, monitor the timing of type II recombination emergence

  • rad27Δ accelerates this process, confirming its role as a negative regulator of telomere recombination

• C-circle Analysis:

  • Measure levels of single-stranded C-rich telomeric DNA circles

  • RAD27 negatively regulates C-circle formation in telomerase-deficient cells

These assays collectively provide a comprehensive view of RAD27's multifaceted roles in maintaining genome integrity through distinct mechanisms . The differential effects observed across these assays help delineate RAD27's specific contributions to various DNA metabolism pathways.

How should researchers interpret conflicting data regarding RAD27's role in mismatch repair?

Interpreting conflicting evidence about RAD27's role in mismatch repair requires nuanced analysis:

The apparently contradictory data can be reconciled through several interpretations:

• Indirect vs. Direct Effects: RAD27 defects might generate substrates that overwhelm the MMR system rather than directly participating in MMR. This would explain why rad27Δ shows some MMR-like phenotypes but also unique mutation signatures .

• Pathway Branching: RAD27 and Exo1 may function in different excision pathways for mismatch repair. While Exo1 serves as the primary exonuclease in MMR, RAD27 might play a role in a specialized branch of MMR, possibly related to particular DNA structures or genomic contexts .

• Cooperative Interaction: RAD27 might facilitate MMR without being a core component, perhaps by processing certain DNA structures that arise during replication which could otherwise interfere with MMR efficiency.

The fact that combining rad27Δ with deletions in MSH2, MLH1, or PMS1 often results in a greater-than-additive increase in mutation rates suggests they affect more than one DNA repair pathway . Similarly, the distinctive pattern of complex mutations in rad27Δ mutants that are flanked by short direct repeats and not observed in MMR mutants indicates a mechanism distinct from classical MMR .

The most parsimonious interpretation is that RAD27 plays a supportive role in genome stability that complements MMR without being a core MMR component, and its loss creates DNA structures that can challenge the MMR machinery.

What are common technical challenges in RAD27 research and how can they be addressed?

Researchers investigating RAD27 should be aware of several technical challenges:

Experimental Challenges and Solutions:

• Temperature Sensitivity Issues:

  • Challenge: rad27Δ shows variable phenotypes at different temperatures

  • Solution: Strictly control temperature during experiments; include internal controls; report exact growth conditions

• Redundant Nuclease Activities:

  • Challenge: Functional redundancy can mask RAD27-specific effects

  • Solution: Use multiple nuclease mutants in combination; employ structure-specific substrates that distinguish different nuclease activities

• Protein Stability Considerations:

  • Challenge: Mutations may affect protein stability rather than specific activity

  • Solution: Verify protein levels by Western blot; include protein stability assays alongside activity measurements

• Secondary Mutation Accumulation:

  • Challenge: rad27Δ strains readily accumulate suppressor mutations due to genomic instability

  • Solution: Generate fresh deletions for each experiment; use multiple independent isolates; sequence verify strains

• Substrate Design Complications:

  • Challenge: In vitro substrates may not accurately reflect physiological complexity

  • Solution: Use multiple substrate designs; include controls for structure formation; validate with in vivo approaches

• Nuclease Contamination:

  • Challenge: Background nuclease activity can confound biochemical assays

  • Solution: Include EDTA controls; use nuclease-free reagents; test heat-inactivated enzyme preparations

• Strain Background Effects:

  • Challenge: Different yeast backgrounds can show different rad27Δ phenotypes

  • Solution: Test multiple backgrounds; document strain lineage; use isogenic strains for comparative analyses

By anticipating these challenges and implementing appropriate controls and solutions, researchers can generate more reliable and reproducible data when studying RAD27 function. The multifaceted nature of RAD27's activities requires careful experimental design to isolate specific functions and accurately interpret results across different experimental systems.