MUS81 Antibody

What is MUS81 and what are its primary cellular functions?

MUS81 is a structure-specific endonuclease belonging to the XPF/MUS81 endonuclease family. It plays critical roles in:

• Resolution of recombination intermediates during DNA repair

• DNA replication during normal cell growth

• Cellular responses to exogenous replicative stress

• Repair after inter-strand cross-links, replication fork collapse, and DNA double-strand breaks

The encoded protein forms a complex with EME1/MMS4 to create a functional endonuclease that resolves Holliday junctions and other branched DNA structures during repair processes.

What is the molecular weight and cellular localization of MUS81?

MUS81 is observed at approximately 70-72 kDa in Western blot applications . It primarily localizes to the nucleus, where it performs its DNA repair functions. Immunofluorescence studies have shown that MUS81 co-localizes with other DNA repair proteins such as RAD51 in the nuclei, particularly after DNA damage induction .

How does MUS81 activity change throughout the cell cycle?

MUS81 activity is temporally regulated during the cell cycle to prevent inappropriate processing of replication intermediates. Research indicates that:

• MUS81-MMS4 endonuclease activity is controlled by cell cycle-dependent phosphorylation

• Activity increases during mitosis when it's needed to resolve persistent recombination intermediates

• During S-phase, MUS81 activity is typically restrained to prevent unscheduled cleavage of replication structures

This temporal regulation ensures cell survival by preventing premature processing of DNA structures essential for replication completion.

How does MUS81 affect DNA replication under normal growth conditions?

MUS81 plays a key role in determining the rate of DNA replication without activating novel replication origins. Studies show that:

• MUS81 promotes replication fork progression

• It reduces the frequency of replication initiation events

• In MUS81-deficient cells, DNA synthesis is slowed

• Cells lacking MUS81 have more frequent replication initiation events

• Despite increased initiation frequency, MUS81-deficient cells use the same pool of replication origins as MUS81-expressing cells

This suggests MUS81 is essential for maintaining normal replication dynamics, even in the absence of exogenous stress.

What happens to DNA replication when MUS81 is depleted?

When MUS81 is depleted (either acutely or chronically):

• DNA synthesis occurs at a significantly slower rate

• Inter-origin distances become shorter, indicating increased frequency of replication initiation events

• There is no significant increase in replication fork asymmetry, suggesting slower synthesis rather than increased fork stalling

• Replication forks are more vulnerable to collapse when exposed to replication inhibitors

• Cells show dependency on the XPF endonuclease for viability

These effects occur rapidly after MUS81 depletion, indicating that MUS81 is continuously involved in determining replication rates rather than representing an adaptive response.

How does MUS81 respond to replication stress?

In response to replication stress, MUS81:

• Facilitates the deceleration of DNA replication after exposure to low doses of replication inhibitors (like aphidicolin)

• Prevents irreversible inhibition of DNA replication

• Creates transient DNA breaks that promote cell survival by facilitating recovery of perturbed replication forks

• Requires its endonuclease activity for these functions

During aphidicolin treatment, MUS81-proficient cells continue replicating at a slow pace, whereas MUS81-deficient cells fully arrest replication, demonstrating MUS81's critical role in managing replication stress.

What are the optimal conditions for using MUS81 antibody in Western blot applications?

For Western blot applications using MUS81 antibody:

• Use affinity-purified polyclonal antibodies (such as rabbit anti-MUS81)

• Expect to observe bands at approximately 70-72 kDa

• Store undiluted antibody at 2-8°C for up to a week for continuous use

• For long-term storage, aliquot and store at -20°C or below

• Avoid repeated freeze/thaw cycles

• Gently mix the antibody solution before use

• Determine optimal dilutions empirically for your specific application and cell type

When validating MUS81 knockdown or knockout, Western blot provides clear confirmation of reduced protein levels.

How can MUS81 antibodies be used to study protein-protein interactions?

MUS81 antibodies can be used to study protein-protein interactions through several techniques:

• Co-immunoprecipitation:

  • MUS81 has been shown to interact with multiple proteins including EME1/MMS4, TRF2, RAD51, and BM28

  • Include appropriate controls (such as DNase I treatment) to exclude bridging effects of nucleic acids

  • Use specific mutants (e.g., TRF2ΔB) to confirm specificity of associations

• Protein Array Analysis:

  • Cell Cycle AntibodyArray™ has been used to detect interactions between MUS81 and proteins involved in cell cycle progression

  • This approach revealed interactions with CyclinB, BM28, BRCA1, BRCA2, and Nibrin proteins

• Mass Spectrometry:

  • For unbiased identification of MUS81-interacting partners, stable cell lines expressing tagged MUS81 can be generated

  • After purification using tag-based chromatography, MUS81 immunoprecipitates can be analyzed by LC-MS/MS

  • This approach identified EME1 and TRF2 as MUS81 binding partners

What controls should be included when using MUS81 antibody for immunofluorescence studies?

When using MUS81 antibody for immunofluorescence studies, include the following controls:

• Positive controls: Cells known to express MUS81 (most proliferating cells)

• Negative controls:

  • Primary antibody omission

  • MUS81 knockdown or knockout cells

  • Isotype control antibody

• Specificity controls:

  • DNase treatment to rule out DNA-mediated co-localization

  • Validation of nuclear localization with DAPI counterstain

• Co-localization controls:

  • Include antibodies against known interacting partners (like RAD51)

  • Use appropriate fluorophore combinations that minimize spectral overlap

A proper co-localization study should include quantification using correlation coefficients (e.g., Pearson's or Mander's) to statistically validate the observed co-localization.

How is MUS81 involved in cancer progression and treatment resistance?

MUS81 plays multiple roles in cancer:

• In serous ovarian cancer (SOC):

  • Elevated MUS81 expression is associated with SOC progression

  • Abnormal expression correlates with poorer clinical outcomes

  • MUS81 participates in genomic instability characteristic of SOC progression

• In gastric cancer:

  • MUS81 negatively correlates with WEE1 expression

  • MUS81 regulates the ubiquitination of WEE1 via E-3 ligase β-TRCP

  • Targeting MUS81 enhances the anticancer effect of WEE1 inhibitor MK1775

• In treatment resistance:

  • Phosphorylation status of MUS81 modifies sensitivity to PARP inhibitors like Olaparib

  • Deregulated function of MUS81/EME1 complex in S-phase can revert Olaparib sensitivity in BRCA2-deficient backgrounds

  • This occurs through cleavage of stalled forks before fork reversal and degradation, followed by Polθ-dependent repair of double-strand breaks

How does MUS81 contribute to telomere maintenance in cancer cells?

In telomerase-negative cancer cells using the Alternative Lengthening of Telomeres (ALT) pathway:

• MUS81 is essential for ALT cell proliferation

• Depletion of MUS81 causes growth arrest in ALT cells within 3-4 weeks

• MUS81 knockdown dramatically induces cell growth arrest in ALT cell lines (GM847, U2OS, SAOS-2)

• The effect is specific for ALT cells, as non-ALT cells show only moderate decrease in viability

• MUS81 interacts with telomere-binding protein TRF2, and this interaction is not mediated by DNA

• The interaction is specific for full-length TRF2, not with TRF2ΔB (a dominant negative mutant lacking the N-terminal domain)

This indicates MUS81 plays a key role in the ALT pathway used by approximately 10-15% of cancers to maintain telomeres in the absence of telomerase.

What strategies can be used to target MUS81 in cancer therapy?

Several approaches for targeting MUS81 in cancer therapy have been investigated:

• Synthetic lethality approaches:

  • MUS81 inhibition enhances sensitivity to PARP inhibitors like Olaparib

  • Combined inhibition of MUS81 and RAD51 induces significant apoptosis (~70%) in SOC cells

• Combination with immune checkpoint inhibitors:

  • MUS81 inhibition increases the accumulation of cytosolic DNA induced by WEE1 inhibitor treatment

  • This activates the DNA sensor STING-mediated innate immunity

  • WEE1 inhibitors specifically enhance the anticancer effect of immune checkpoint blockade therapy in MUS81-deficient gastric cancer cells

• Targeting ALT-dependent cancers:

  • MUS81 inhibition specifically affects the viability of ALT cells

  • This offers a selective therapeutic opportunity for telomerase-negative cancers

These approaches highlight MUS81 as a promising target for precision oncology, especially in combination therapy strategies.

How can contradictory data about MUS81 function be reconciled in experimental design?

Researchers have noted apparently contradictory data regarding MUS81 function, particularly in response to replication stress. To reconcile these contradictions:

• Consider cell-type specific differences:

  • Different cell types may have varying dependencies on MUS81

  • Different genetic backgrounds may compensate for MUS81 deficiency differently

• Distinguish between acute and chronic MUS81 depletion:

  • Acute knockdown by siRNA may produce different phenotypes than stable knockout

  • Studies show both acute and chronic depletion slow DNA synthesis, but adaptations may differ

• Evaluate checkpoint activation status:

  • Monitor checkpoint proteins like Rad53 phosphorylation

  • Assess spindle elongation in S-phase cells

  • Confirm checkpoint proficiency in MUS81-deficient cells

• Use complementary assays:

  • Combine bulk DNA replication assays with more sensitive methods like DNA combing

  • PFGE can detect completion of chromosome replication

  • DNA fiber analysis provides direct visualization of ongoing replication

A systematic approach using multiple experimental techniques helps resolve contradictions in MUS81 function data.

What methodological approaches can reveal MUS81's role in replication fork dynamics?

Advanced methodologies to study MUS81's role in replication fork dynamics include:

• DNA fiber analysis:

  • Sequential labeling with nucleotide analogs (IdU/CldU)

  • Measures rates of replication fork progression

  • Detects replication track asymmetry as a marker of fork stalling

  • Calculate fork density per Mb DNA fiber

• Density-shift experiments:

  • Analyze replication of specific genomic regions

  • Track the progression of replication forks through multiple fragments of a replicon

  • Compare percentage of replicated DNA in different fragments at various time points

• Cell synchronization techniques:

  • Double thymidine block to synchronize at G1/S boundary

  • Additional Hoechst 33342 block to enrich cells at G2 phase

  • Methionine restriction for cell cycle control

These approaches provide complementary data on how MUS81 influences replication dynamics at both global and locus-specific levels.

What factors should be considered when analyzing MUS81 phosphorylation status and its effect on enzyme activity?

When studying MUS81 phosphorylation:

• Cell cycle phase specificity:

  • MUS81 phosphorylation status varies throughout the cell cycle

  • Mitotic phosphorylation activates the complex

  • S-phase phosphorylation may inhibit premature activation

• Kinase identification:

  • Multiple kinases may phosphorylate MUS81 at different residues

  • CDKs, Plks, and checkpoint kinases have been implicated

  • Site-specific mutants can determine functional relevance

• Techniques for phosphorylation detection:

  • Phospho-specific antibodies

  • Mass spectrometry for unbiased phosphorylation site mapping

  • Mobility shift assays (phosphorylated proteins often migrate differently on SDS-PAGE)

• Functional consequence assessment:

  • Endonuclease activity assays using purified proteins

  • Cell-based assays for DNA repair efficiency

  • Resistance to DNA-damaging agents like Olaparib

Understanding phosphorylation-dependent regulation of MUS81 is critical for designing targeted therapeutic approaches, especially in the context of cancer treatment resistance mechanisms.