Recombinant Poly (ADP-ribose) glycohydrolase pme-3 (pme-3), partial

What is the functional role of PARG-1/pme-3 in C. elegans meiosis?

PARG-1/pme-3 in C. elegans has revealed an unexpected role beyond its canonical function in poly(ADP-ribose) catabolism. Studies have demonstrated that PARG-1 is involved in coordinating meiotic DNA double-strand break (DSB) formation and homologous recombination-mediated repair . This function appears to be independent of its catalytic activity in poly(ADP-ribose) hydrolysis.

Specifically, PARG-1 promotes:

• The induction of meiotic DNA breaks

• Homologous recombination-mediated repair of these breaks

• Regulation of crossover numbers and distribution (shaping the recombination landscape)

The critical nature of PARG-1 is evidenced by repair defects observed in mutants. When PARG-1 function is compromised alongside mutations in other DNA repair genes, such as mre-11, repair efficiency is significantly reduced, suggesting that PARG-1 influences DNA repair pathway choice .

How does PARG-1/pme-3 interact with the meiotic machinery in C. elegans?

PARG-1/pme-3 interacts with multiple components of the meiotic machinery to facilitate proper DSB formation and repair:

Protein Interaction Network:

• Synaptonemal complex components: PARG-1 associates with both axial and central elements of the synaptonemal complex

• Cohesins: Interacts with REC-8/Rec8, a meiosis-specific cohesin component

• MRN/X complex: Co-immunoprecipitation studies revealed interaction with MRE-11, a component of the MRN/X complex involved in DSB processing

• HTP-3: Interacts with this axial element component which is known to promote meiotic DSB formation

These interactions position PARG-1 within critical meiotic protein assemblies. Co-immunoprecipitation experiments with MRE-11::GFP have confirmed the physical association between PARG-1 and MRE-11, suggesting a molecular mechanism by which PARG-1 might influence DSB formation . The interaction with HTP-3, which itself interacts with MRE-11, provides additional support for PARG-1's involvement in a pro-DSB formation pathway that operates parallel to other known pathways involving factors like HIM-17, HIM-5, and DSB-2 .

What are the biochemical properties of PARG-1/pme-3 and how does it catalyze poly(ADP-ribose) hydrolysis?

PARG-1/pme-3, like other PARGs, catalyzes the hydrolysis of glycosidic bonds of ADP-ribose polymers. Its biochemical activity includes:

Catalytic Activities:

• Endoglycosidase activity: Cleaves internal glycosidic bonds within PAR chains, releasing free PAR fragments

• Exoglycosidase activity: Preferentially binds to the two most distal ADP-ribose residues and cleaves from the ends of PAR chains, releasing mono ADP-ribose units

• Enzymatic classification: EC 3.2.1.143 (glycoside hydrolase)

The catalytic process specifically targets α(1″–2′) or α(1″′–2″) glycosidic linkages in the PAR polymer . While the exoglycosidase activity predominates under normal conditions, the endoglycosidase activity becomes more important during hyper-PARP activation, such as during DNA damage responses .

The released ADP-ribose monomers are subsequently metabolized into AMP and ribose 5′ phosphate by ADP-ribose pyrophosphohydrolases like the NUDIX family enzymes . These metabolites then feed into various cellular pathways:

• AMP is utilized in ATP reformation and cell signaling pathways

• Ribose 5′ phosphate serves as a precursor for biomolecules including DNA, RNA, and ATP

What methods can researchers use to express and purify recombinant PARG-1/pme-3?

Researchers working with recombinant PARG-1/pme-3 should consider the following methodological approaches:

Expression System:

• E. coli expression: Both commercial sources and published protocols indicate successful expression in E. coli systems

• Partial vs. full-length protein: Consider whether full-length or partial constructs are appropriate for your research question. Commercial sources offer partial constructs that may have better stability or expression yields

Purification and Storage Recommendations:

• Typical purity: >85% as assessed by SDS-PAGE for research-grade preparations

• Reconstitution: Centrifuge vials briefly before opening and reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Storage buffer: Addition of glycerol (5-50% final concentration) is recommended for long-term storage

• Storage temperature: -20°C/-80°C is optimal, with shelf life of approximately 12 months for lyophilized preparations and 6 months for liquid preparations

• Working aliquots: Can be stored at 4°C for up to one week

• Stability considerations: Repeated freeze-thaw cycles should be avoided

How does PARG-1/pme-3 contribute to the regulation of meiotic DNA double-strand breaks?

PARG-1/pme-3 appears to regulate meiotic DSBs through multiple mechanisms:

DSB Formation Pathway:

• Parallel pathway operation: PARG-1 operates in a pathway distinct from but parallel to the pathway involving HIM-17, HIM-5, and DSB-2

• Interaction with pro-DSB factors: The HTP-3-MRE-11 axis is a key interaction point for PARG-1's pro-DSB function

Experimental Evidence:

Analysis of mutant phenotypes provides insights into PARG-1's role:

• Synergistic effects: When parg-1 mutations are combined with mutations in him-17, him-5, or dsb-2, synergistic defects in DSB formation are observed, confirming PARG-1's operation in a parallel pathway

• Protein localization: Expression and localization studies show that PARG-1 and the HIM factors (HIM-17, HIM-5, or DSB-2) are not mutually dependent, further supporting the parallel pathway model

While the precise mechanism of PARG-1's contribution to DSB formation remains under investigation, current evidence suggests it works through protein-protein interactions rather than by direct regulation of SPO-11 (the endonuclease that creates meiotic DSBs). This is supported by the observation that parg-1 mutants do not show the severe defects in bivalent formation or RAD-51 loading that would be expected if SPO-11 recruitment were compromised .

What is the relationship between PARG-1/pme-3 and PARP enzymes in C. elegans?

C. elegans possesses both poly(ADP-ribose) polymerases (PARPs) and PARG enzymes that work together to regulate poly(ADP-ribosyl)ation:

elegans PARP/PARG System:

• PME-1: 108 kDa protein with 31% identity to human PARP-1, contains zinc-finger motifs similar to other PARP-1 subfamily members

• PME-2: 61 kDa protein with 24% identity to human PARP-2

• PARG-1/PME-3: The poly(ADP-ribose) glycohydrolase that reverses PARP activity

These enzymes coordinate a balanced cycle of poly(ADP-ribosyl)ation:

• PME-1 and PME-2 catalyze the addition of ADP-ribose units to target proteins

• PARG-1/PME-3 removes these modifications by hydrolyzing the glycosidic bonds

Studies have shown that recombinant PME-1 and PME-2 display PARP activity that accounts for the poly(ADP-ribosyl)ation observed in C. elegans extracts . The expression patterns of pme-1 and pme-2 are developmentally regulated, suggesting stage-specific functions .

Interestingly, mRNA for pme-1 is 5'-tagged with splice leader 1, whereas mRNA for pme-2 is tagged with splice leader 2, suggesting an operon-like expression pattern for pme-2 . This organization may have implications for the coordinated regulation of these enzymes.

What methods can researchers use to analyze PARG-1/pme-3 activity in experimental settings?

Researchers investigating PARG-1/pme-3 can employ several methodological approaches:

In vitro Activity Assays:

• Glycohydrolase activity measurement: Commercial assay kits are available that can be adapted for PARG-1 activity measurement, similar to those used for PARP3

• Substrate preparation: Purified PAR polymers of defined length can be used as substrates

• Product detection: Released ADP-ribose monomers can be quantified through coupled enzymatic reactions or chromatographic methods

In vivo Functional Analysis:

• Genetic approaches: Analysis of single and double mutants to assess genetic interactions (e.g., parg-1 with mre-11 or com-1)

• Irradiation assays: Comparing DNA repair efficiency in wild-type vs. parg-1 mutants after irradiation

• Immunofluorescence microscopy: Monitoring markers of DSB formation and repair (e.g., RAD-51) in germline nuclei

Protein Interaction Studies:

• Co-immunoprecipitation: Using tagged versions of PARG-1 or potential interacting partners (e.g., MRE-11::GFP) to identify physical associations

• Yeast two-hybrid: Screening for additional interaction partners

• Proximity ligation assays: Visualizing in situ protein interactions in intact cells or tissues

How do mutations in PARG-1/pme-3 affect meiotic recombination and DNA repair?

Mutations in PARG-1/pme-3 have specific effects on meiotic processes:

Phenotypic Consequences:

• Crossover regulation: PARG-1 influences the tightly regulated control of crossover numbers and distribution along chromosomes

• DSB repair pathway choice: PARG-1 affects DNA repair pathway choice when certain other repair factors (e.g., MRE-11) are compromised

Experimental Observations:

• Synthetic interactions: When parg-1 mutations are combined with mutations in other DSB-promoting genes, synergistic defects in DSB formation occur

• Irradiation response: In irradiation experiments, parg-1; mre-11 double mutants show repair deficiencies compared to non-irradiated controls, suggesting PARG-1 influences repair pathway choice specifically when MRE-11 function is compromised

• Contrast with other pathways: Interestingly, parg-1; com-1 double mutants don't show the same deficiency, indicating pathway specificity

These findings indicate that PARG-1 has specialized roles in both promoting DSB formation and influencing their repair, with effects that become particularly evident in certain genetic backgrounds.

What are the structural characteristics of PARG-1/pme-3 that distinguish it from mammalian PARG homologs?

While detailed structural information specific to C. elegans PARG-1/pme-3 is limited in the provided search results, we can infer some characteristics:

Structural Features:

• Catalytic domain: Contains the core enzymatic region responsible for glycohydrolase activity (EC 3.2.1.143)

• Conserved function: Despite evolutionary distance from mammalian PARGs, C. elegans PARG-1 retains the fundamental ability to hydrolyze PAR polymers

• Functional regions: May contain both catalytic and protein interaction domains, given its dual roles in catalysis and meiotic protein interactions

Recombinant PARG-1/pme-3 proteins are available commercially in partial forms , suggesting that certain domains may be more amenable to expression and purification than others.

The research indicates that PARG-1's function in meiosis does not require its catalytic activity , pointing to the importance of non-catalytic domains in mediating protein-protein interactions with components of the meiotic machinery.

How can researchers investigate the non-catalytic functions of PARG-1/pme-3?

Since PARG-1/pme-3 has functions beyond its enzymatic activity, specific approaches can help dissect these roles:

Methodological Approaches:

• Catalytically inactive mutants: Generate point mutations in the catalytic domain that abolish enzymatic activity while preserving protein structure

• Domain deletion/mutation analysis: Create constructs lacking specific domains or containing mutations in interaction surfaces

• Structure-function analysis: Compare phenotypes of various mutant forms to identify regions required for specific functions

Experimental Design Strategy:

• Genetic separation of functions: Test whether catalytically inactive PARG-1 can rescue meiotic defects in parg-1 null mutants

• Protein interaction mapping: Identify which domains of PARG-1 mediate interactions with meiotic proteins like HTP-3 and MRE-11

• In vivo localization: Determine if catalytically inactive PARG-1 localizes properly to meiotic chromosomes

The finding that PARG-1 shapes the recombination landscape and regulates crossover numbers without requiring its catalytic activity provides a foundation for these investigations. Creating separation-of-function mutants would help further dissect the enzymatic versus structural roles of this protein in meiosis.