Recombinant Xenopus laevis E3 SUMO-protein ligase NSE2 (nsmce2)

What is the function of NSMCE2 in Xenopus laevis and how is it characterized?

NSMCE2 (also known as MMS21) functions as an essential SUMO ligase component of the SMC5/6 complex in Xenopus. Its primary roles include:

• DNA repair, particularly double-strand break repair via homologous recombination

• Chromosome segregation during cell division

• Suppression of inappropriate recombination

• Maintenance of genomic stability

The protein contains a C-terminal SP-RING domain with E3 SUMO ligase activity required for DNA damage repair . NSMCE2 can be characterized through:

• Western blot analysis using antibodies against NSMCE2

• Immunofluorescence to detect localization patterns

• Recombinant expression and purification for in vitro activity assays

• Gene knockout or knockdown approaches using morpholinos or CRISPR-Cas9

In mouse models, NSMCE2 deficiency causes early embryonic lethality due to segregation abnormalities, suggesting it plays similarly critical roles in early Xenopus development .

What are the optimal methods for recombinant expression of Xenopus laevis NSMCE2?

For successful recombinant expression of Xenopus laevis NSMCE2, researchers should consider:

Expression Systems:

• E. coli using pET or pGEX vectors for GST-fusion proteins

• Baculovirus expression for more native-like post-translational modifications

• Cell-free Xenopus egg extracts for expression in a native environment

Purification Strategy:

• Affinity chromatography using His6, GST, or MBP tags

• Ion exchange chromatography for further purification

• Size exclusion chromatography for final polishing

Optimization Considerations:

• Codon optimization for the expression system

• Lower expression temperatures (16-18°C) to improve solubility

• Co-expression with chaperones if solubility is an issue

• Expression of specific domains (e.g., SUMO ligase domain) if full-length protein is difficult to express

Quality Control:

• In vitro SUMOylation assays to confirm E3 ligase activity

• Binding assays with SMC5 to verify complex formation capability

Based on research with human NSMCE2, recombinant full-length protein fused to GST has been successfully used to generate antibodies , suggesting this is a viable expression strategy.

What assays can be used to measure NSMCE2 SUMO ligase activity in vitro?

To measure NSMCE2 SUMO ligase activity, researchers can employ several complementary approaches:

In vitro SUMOylation Assay:

• Components: Purified E1 enzyme (SAE1/SAE2), E2 enzyme (UBC9), SUMO protein, ATP, and substrate protein

• Recombinant NSMCE2 is added to test its ability to enhance SUMOylation

• Detection by Western blot showing higher molecular weight band of SUMOylated substrate

SUMO Chain Formation Assay:

• Similar to SUMOylation assay but focused on NSMCE2's ability to promote SUMO chain formation

• Detection of high molecular weight SUMO chains by Western blot

ATP Dependency Testing:

• Compare SUMOylation efficiency with and without ATP

• Include SMC5 protein as NSMCE2 activity depends on ATP binding to SMC5

Table 1: Components Required for In Vitro NSMCE2 SUMOylation Assay

From the search results, the SUMOylation activity of NSMCE2 depends on ATP binding to SMC5 , suggesting that in vitro assays should include both proteins to observe physiologically relevant activity.

How does the SUMO pathway function during Xenopus development?

The SUMO pathway plays crucial roles during Xenopus development:

SUMO Machinery Components:

• SUMO proteins (synthesized as precursors requiring maturation)

• E1 activating enzyme: heterodimeric SAE1/SAE2

• E2 conjugating enzyme: UBC9

• E3 ligases: PIAS family proteins, NSMCE2, and others

• DeSUMOylating enzymes: SENP family proteins

Developmental Functions:

• Neural plate cell apical constriction and neural tube closure require folate receptor-1

• Retinal progenitor proliferation involves Sp1 SUMOylation in Xenopus

• PIAS3-mediated SUMOylation of Nr2e3 regulates rod photoreceptor specification

Localization Patterns:
During oogenesis and early development, SUMO proteins show distinct localization patterns:

• In transcriptionally active oocytes: nucleoplasm and chromatin

• During cell division: spindle poles, centromeres, and separating chromosomes

Research Methods:

• Morpholino knockdown of SUMO pathway components

• Expression of dominant-negative or constitutively active SUMO pathway mutants

• CRISPR-Cas9 gene editing of pathway components

• Transgenic expression of tagged SUMO for visualization

The SUMO pathway works in coordination with the ubiquitin-proteasome system during developmental transitions , making it an important regulatory mechanism during Xenopus embryogenesis.

How does NSMCE2 contribute to chromosome segregation during early Xenopus embryogenesis?

NSMCE2 plays a critical role in chromosome segregation during early Xenopus embryogenesis, particularly during the rapid cell divisions characteristic of amphibian development:

Mechanisms of Action:

• Resolution of topological DNA entanglements before chromosome separation

• SUMOylation of factors involved in chromosome dynamics

• Ensuring proper centromere and kinetochore function

• Facilitating removal of cohesion between sister chromatids

Phenotypes of NSMCE2 Deficiency:
Based on mouse studies, NSMCE2 deficiency in Xenopus likely causes:

• Irregular nuclear size and shape

• Multilobulated nuclei

• Aberrant chromosome condensation

• Failed chromosome segregation leading to developmental arrest

Experimental Approaches:

• Time-lapse microscopy of dividing cells in NSMCE2-depleted embryos

• Visualization of mitotic chromosomes using fluorescent histone markers

• Analysis of spindle organization using tubulin immunostaining

• Chromosome spread preparations to directly observe segregation defects

The large cells of early Xenopus embryos provide an excellent system for high-resolution imaging of chromosome dynamics, offering advantages over mammalian models for studying segregation defects. NSMCE2 heterozygous cells show higher incidence of micronuclei and polynucleated cells , indicating that even partial loss of function impacts chromosome segregation.

What is the impact of NSMCE2 deficiency on homologous recombination in Xenopus?

NSMCE2 deficiency significantly impacts homologous recombination (HR) in Xenopus based on findings from other model systems:

Observed Effects on Recombination:

• Increased rates of spontaneous sister chromatid exchanges (SCEs)

• Elevated inter-telomeric recombination (T-SCE)

• Accumulation of BRCA1 foci, indicative of increased recombination activity

• MUS81 nuclease-dependent increases in recombination

Molecular Mechanisms:

• NSMCE2 normally suppresses inappropriate recombination

• Without NSMCE2, cells show accumulation of recombination intermediates

• These intermediates require resolution by structure-specific nucleases like MUS81

Experimental Methods for Studying HR Defects:

• SCE assays in cells derived from NSMCE2-depleted embryos

• Immunostaining for HR markers (RAD51, BRCA1) after DNA damage

• Comet assays to assess DNA break accumulation

• FISH-based approaches to measure telomere recombination

Table 2: Comparison of Recombination Phenotypes with and without NSMCE2

From the search results, NSMCE2-deficient cells show increased rates of SCEs even without exogenous DNA damaging agents , indicating that NSMCE2 plays a constitutive role in suppressing spontaneous recombination during normal DNA metabolism.

How is ATP-dependent regulation of NSMCE2 SUMO ligase activity mediated?

The ATP-dependent regulation of NSMCE2 SUMO ligase activity represents a sophisticated control mechanism:

Structural Basis:

• Despite physical distance between ATP-binding and SUMO ligase domains, communication occurs

• A conserved disruption in SMC5's coiled-coil domain enables this communication

• ATP binding to SMC5 is required for NSMCE2-dependent SUMOylation

Molecular Mechanism:

• ATP binding induces conformational changes in the SMC5/6 complex

• These changes physically remodel the SMC5-NSMCE2 heterodimer

• Remodeling positions NSMCE2 optimally for interaction with UBC9 and substrate proteins

• Scanning force microscopy confirms ATP-dependent physical remodeling

Functional Significance:

• Ensures SUMOylation only occurs in context of active SMC5/6 complex

• Coordinates ATP-dependent DNA manipulation with protein modification

• Prevents inappropriate SUMOylation of targets

Experimental Approaches:

• In vitro SUMOylation assays with wild-type vs. ATP-binding mutants

• Structural studies of the SMC5-NSMCE2 complex

• FRET-based approaches to detect conformational changes

• Mutagenesis of the coiled-coil disruption region

This ATP-dependent regulation mechanism reveals that "the ATP-binding activity of the Smc5/6 complex is coordinated with its SUMO ligase, through the coiled coil domain of Smc5 and the physical remodeling of the molecule, to promote sumoylation and chromosome disjunction during DNA repair" .

How does NSMCE2's SUMO ligase activity differ from its structural role in the SMC5/6 complex?

NSMCE2 exhibits dual functionality through its SUMO ligase activity and structural role in the SMC5/6 complex:

SUMO Ligase Activity:

• Mediated by the C-terminal SP-RING domain

• Catalyzes transfer of SUMO from UBC9 to substrate proteins

• Regulated by ATP binding to SMC5

• Can be experimentally disrupted by point mutations (e.g., C195S and H197A)

Structural Role:

• N-terminal domain docks to the arm region of SMC5

• Required for proper complex assembly and stability

• Potentially serves as a binding platform for other proteins

• Functions independently of SUMO ligase activity

Evidence for Separable Functions:
From the research in mice:

• NSMCE2's SUMO ligase activity is dispensable for viability (NSMCE2 SD mutant mice)

• Complete NSMCE2 deletion causes embryonic lethality

• This demonstrates that NSMCE2's structural role is essential

Experimental Approaches to Distinguish Functions:

• Express SUMO ligase-dead mutants (e.g., NSMCE2 SD) in Xenopus

• Compare phenotypes with complete NSMCE2 knockout

• Identify functions dependent on catalytic activity versus protein presence

• Analyze chromosome segregation and recombination in each condition

The search results reveal that "SUMO- and BLM-independent activities of NSMCE2 limit recombination and facilitate segregation; functions of the SMC5/6 complex that are necessary to prevent cancer and aging in mice" , highlighting the importance of NSMCE2's structural role beyond its enzymatic activity.

How can researchers design experiments to investigate NSMCE2's role in DNA repair pathways in Xenopus?

Designing rigorous experiments to investigate NSMCE2's role in DNA repair requires leveraging Xenopus-specific advantages:

Cell-Free Extract System Approach:

• Prepare high-speed extracts from Xenopus eggs

• Immunodeplete endogenous NSMCE2

• Add back recombinant wild-type or mutant NSMCE2

• Introduce DNA templates with specific damages (DSBs, crosslinks, etc.)

• Monitor repair efficiency using plasmid replication/repair assays

In Vivo Embryo Approach:

• Knockdown/knockout NSMCE2 using morpholinos or CRISPR-Cas9

• Confirm knockdown/knockout efficiency by Western blot

• Expose embryos to DNA damaging agents (UV, IR, chemical mutagens)

• Assess:

  • Survival rates

  • Developmental defects

  • DNA repair marker recruitment (γH2AX, 53BP1, RAD51)

Pathway-Specific Analysis:

• Homologous recombination: RAD51 foci formation, DR-GFP reporter

• Non-homologous end joining: DNA ligase IV activity, EJ5-GFP reporter

• Interstrand crosslink repair: Sensitivity to mitomycin C, FANCD2 monoubiquitination

Table 3: Experimental Design for NSMCE2's Role in Different Repair Pathways

From the search results, NSMCE2 deficiency leads to increased BRCA1 foci , providing a starting point for investigating its specific roles in homologous recombination. The increased SCE rates in NSMCE2-deficient cells depend on MUS81 nuclease , suggesting a specific mechanism to explore in Xenopus.

What methods can be used to study the interplay between NSMCE2 and BLM helicase in Xenopus?

The relationship between NSMCE2 and BLM helicase represents a critical intersection in DNA metabolism regulation that can be investigated in Xenopus:

Background:

• In yeast, smc5/6 mutants phenocopy mutations in sgs1 (BLM ortholog)

• NSMCE2 deletion in mice leads to pathologies resembling Bloom's syndrome

• Despite similarities, NSMCE2 and BLM foci do not colocalize

• Concomitant deletion of Blm and Nsmce2 increases recombination and is synthetic lethal

Experimental Approaches:

1. Genetic Interaction Studies:

• Generate single and double knockdowns/knockouts in Xenopus

• Compare phenotypic severity between single and double manipulations

• Assess synthetic lethality or enhancement

2. Cytological Analysis:

• Immunofluorescence to visualize NSMCE2 and BLM localization

• Quantify SCEs in single and double mutants

• Analyze chromosome mis-segregation frequency

3. Biochemical Approaches:

• Immunoprecipitation to detect potential physical interactions

• Mass spectrometry to identify shared interaction partners

• In vitro reconstitution of recombination intermediates processing

4. Functional Assays:

• Design assays to detect recombination intermediate accumulation

• Monitor dissolution of double Holliday junctions

• Analyze telomere maintenance in single and double mutants

Table 4: Comparison of NSMCE2 and BLM Deficiency Effects

From the search results, "concomitant deletion of Blm and Nsmce2 in B lymphocytes further increases recombination rates and is synthetic lethal due to severe chromosome mis-segregation" . This synthetic lethality provides a powerful framework for investigating how these two proteins function in parallel pathways to maintain genome stability in Xenopus.

How can the role of NSMCE2 in telomere maintenance be investigated in Xenopus?

Investigating NSMCE2's role in telomere maintenance in Xenopus requires specialized approaches:

Background:

Experimental Strategies:

1. Telomere Length Analysis:

• Prepare genomic DNA from control and NSMCE2-depleted Xenopus cells/tissues

• Perform terminal restriction fragment (TRF) analysis

• Use quantitative FISH with telomere-specific probes

• Employ single-telomere length analysis (STELA) for individual telomeres

2. Telomere Recombination Assessment:

• CO-FISH (Chromosome Orientation FISH) to detect T-SCEs

• 2D gel electrophoresis to detect telomeric recombination intermediates

• Analysis of telomere circles as indicators of alternative lengthening of telomeres (ALT)

3. Shelterin Complex Analysis:

• Immunoprecipitate telomeric proteins (TRF1, TRF2, POT1)

• Assess SUMOylation status with and without NSMCE2

• Identify specific SUMOylation sites by mass spectrometry

• Generate SUMOylation-deficient mutants of telomeric proteins

4. Telomere Maintenance Pathway Investigation:

• Analyze telomerase activity using TRAP assay

• Assess ALT markers (APBs, C-circles) in NSMCE2-deficient cells

• Examine telomere fragility after replication stress

Table 5: Methods for Analyzing Telomere Phenotypes in NSMCE2-Deficient Xenopus

How can researchers design experiments to analyze the effects of NSMCE2 mutations identified in human disease using Xenopus models?

Xenopus provides an excellent model to investigate the developmental consequences of disease-associated NSMCE2 mutations:

Background:

• Human NSMCE2 mutations are linked to primordial dwarfism, extreme insulin resistance, and gonadal failure

• Two patients with compound heterozygous frameshift mutations (p.Ser116Leufs18 and p.Ala234Glufs4) have been identified

• These mutations likely cause hypomorphic (reduced function) rather than complete loss-of-function

Experimental Design:

1. Generation of Disease-Relevant Models:

• CRISPR-Cas9 gene editing to introduce equivalent mutations in Xenopus nsmce2

• Morpholino knockdown with co-injection of mutant mRNA for rescue experiments

• Generation of transgenic lines expressing human mutant NSMCE2

2. Developmental Phenotyping:

• Growth measurements throughout development

• Skeletal development assessment (cartilage/bone staining)

• Gonadal development analysis

• Insulin signaling pathway evaluation

3. Molecular Characterization:

• Quantify mutant NSMCE2 expression levels

• Assess SUMO ligase activity of mutant proteins

• Analyze SUMOylation patterns in affected tissues

• Evaluate DNA damage response and repair capacity

4. Tissue-Specific Effects:

• Focus on tissues affected in human patients:

  • Skeletal system (primordial dwarfism)

  • Pancreas/metabolic tissues (insulin resistance)

  • Gonads (reproductive failure)

Table 6: Experimental Approach to Model Human NSMCE2 Mutations in Xenopus

The large size, external development, and manipulability of Xenopus embryos make them particularly well-suited for studying the developmental impact of disease-associated mutations. By focusing on equivalent mutations to those found in human patients, researchers can gain insights into the pathophysiological mechanisms underlying NSMCE2-associated disorders.