Recombinant Xenopus laevis DNA topoisomerase 2-binding protein 1-B (topbp1-B), partial

What is the functional role of TopBP1 in Xenopus laevis DNA replication?

TopBP1 serves as a critical scaffold protein in Xenopus laevis, playing dual roles in DNA replication initiation and regulation of checkpoint responses. TopBP1 is essential for the initiation of DNA replication through its interaction with several replication factors and its ability to facilitate the loading of Cdc45 onto replication origins . In Xenopus egg extracts, TopBP1 collaborates with Treslin in a Cdk2-dependent manner to regulate a pivotal step in DNA replication initiation . The protein contains multiple BRCT (BRCA1 C-Terminal) domains that mediate its interactions with various protein partners involved in replication .

Experimental evidence shows that the N-terminal region of TopBP1 containing BRCT domains I-III is both necessary and sufficient for DNA replication in Xenopus egg extracts . Depletion of TopBP1 from egg extracts dramatically inhibits chromosomal DNA replication, but this can be rescued by adding back recombinant TopBP1 fragments containing BRCT repeats I-III . This indicates that this region of TopBP1 is critical for its replication function.

How does TopBP1 activate ATR kinase in checkpoint responses?

TopBP1 is a key activator of the ATR (ATM and Rad3-related) kinase, which is essential for checkpoint responses to incompletely replicated and damaged DNA. The activation mechanism involves:

• Direct interaction and activation: Recombinant TopBP1 induces a large increase in the kinase activity of both Xenopus and human ATR . This activation is mediated by a specific ATR-activating domain (AAD) that resides in a conserved segment of TopBP1 distinct from its numerous BRCT repeats .

• Recruitment to DNA damage sites: TopBP1 is recruited to sites of DNA damage where it can activate ATR. The BRCT1 and BRCT7 domains are particularly important for this recruitment process, with BRCT1 mediating a phosphorylation-dependent interaction with binding partners at double-strand breaks (DSBs) .

• Downstream signaling: Once activated, ATR phosphorylates downstream targets like Chk1 to trigger checkpoint responses. Experimental data from Xenopus egg extracts shows that mutations in the ATR-activating domain of TopBP1 render the extracts defective in checkpoint regulation .

The isolated ATR-activating domain from TopBP1 can induce ectopic activation of ATR-dependent signaling in both Xenopus egg extracts and human cells, demonstrating the critical nature of this interaction .

What methods can be used to express and purify recombinant Xenopus laevis TopBP1?

For successful expression and purification of recombinant Xenopus laevis TopBP1, researchers typically employ the following methodological approach:

Expression System Selection:

• Baculovirus expression systems are commonly used for full-length TopBP1 due to its large size (~180 kDa) and need for eukaryotic post-translational modifications

• E. coli systems may be suitable for expressing individual BRCT domains or smaller fragments

Purification Protocol:

• Affinity Tags: Add N- or C-terminal tags (His, FLAG, or SFB) to facilitate purification

• Cell Lysis: Lyse cells in buffer containing protease inhibitors and phosphatase inhibitors (to preserve phosphorylation state)

• Affinity Chromatography: Purify using appropriate affinity resin

• Ion Exchange Chromatography: Further purify using anion exchange

• Size Exclusion Chromatography: Final polishing step to isolate homogeneous protein

Quality Control Assessments:

• SDS-PAGE to verify size and purity

• Western blotting to confirm identity

• Functional assays (e.g., ATR activation assay) to verify activity

Recent studies have successfully used FLAG-tagged, truncated versions of TopBP1 containing BRCT domains I-VI for isolation from Xenopus egg extracts and functional studies . For full-length protein, expressing and purifying from insect cells using baculovirus systems has yielded functionally active protein capable of activating ATR .

What are the distinct functions of different BRCT domains in TopBP1?

TopBP1 contains eight or nine BRCT domains, each with specialized functions in DNA replication and checkpoint control. The functional specialization is summarized in the table below:

Research using Xenopus egg extracts has demonstrated that different BRCT domains have distinct roles:

• The N-terminal region (BRCT0-1-2, BRCT3, and GINI region) is sufficient for DNA replication

• BRCT5 plays a role downstream of recruitment in promoting ATR-mediated CHK1 phosphorylation

• BRCT7 has a dual role in recruitment to DSBs and independently in promoting ATR signaling

This functional specialization allows TopBP1 to coordinate multiple nuclear processes through its diverse interaction network.

How can I study TopBP1-mediated ATR activation using Xenopus egg extracts?

The DMAX (DNA-Mediated ATR Activation in Xenopus) system provides a powerful tool for studying TopBP1-mediated ATR signaling in Xenopus egg extracts. This methodology offers several advantages for investigating the biochemical mechanisms of TopBP1 function:

DMAX System Protocol:

• Preparation of DNA Double-Strand Breaks (DSBs):

  • Generate linear dsDNA molecules (optimally 5000 nucleotides) by PCR

  • For isolation experiments, add biotin to one end of the DNA and couple to magnetic streptavidin beads

• Incubation with Xenopus Egg Extract (XEE):

  • Add DNA at femtomolar concentration to XEE

  • Incubate at room temperature to allow ATR activation

• Analysis of ATR Activation:

  • Assess phosphorylation of CHK1 by Western blotting

  • For protein recruitment studies, isolate DNA-bound proteins using magnetic beads, wash, and elute for analysis

• Immunodepletion and Add-Back Experiments:

  • Deplete endogenous TopBP1 using specific antibodies

  • Add back wild-type or mutant recombinant TopBP1 to study domain functions

This system has revealed that DNAs of 5000 nucleotides at femtomolar concentration potently activate ATR . Using immunodepletion and add-back of TopBP1 point mutants, researchers have determined that BRCT1 and BRCT7 are important for recruitment to DSBs, while BRCT5 functions downstream to promote ATR-mediated phosphorylation of CHK1 .

The DMAX system provides several advantages:

• Physiologically relevant DSB-induced activation of ATR

• Ability to isolate proteins bound to DNA breaks

• Capacity to perform structure-function analysis using mutant proteins

What is the mechanism of TopBP1 interaction with GINS in regulating DNA replication?

TopBP1 utilizes a bipartite binding mode to interact with the GINS complex, which is essential for genome replication. This interaction coordinates critical steps in replication origin firing:

Structural Basis of the Interaction:

• TopBP1 interacts with the GINS complex through two separate binding surfaces that bind to opposite ends of the A domain of the GINS subunit Psf1

• The interaction involves both the GINI (GINS interaction) region and the BRCT4 domain of TopBP1

• Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, but with reduced affinity

Functional Significance:

• In Xenopus egg extracts, either binding surface alone is sufficient for replication origin firing, but becomes essential in the absence of the other

• The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45

• This suggests a model where TopBP1 must be ejected for Polε incorporation into the replisome

Experimental Evidence:

• Proximity biotinylation experiments in human cells show enrichment of GINS subunits (Psf3, Psf2) upon APEX2-tagged TopBP1 pulldown

• Chromatin isolation from Xenopus egg extracts demonstrates that origin licensing (Mcm2-7 loading) is independent of TopBP1, but GINS and Cdc45 loading onto chromatin requires TopBP1

• Yeast two-hybrid experiments showed interaction of human and Xenopus TopBP1-GINI region with GINS (Psf1 and Psf3)

The TopBP1-GINS interaction model provides insights into how three molecular processes are coordinated during origin firing: DNA polymerase epsilon arrival, TopBP1 ejection, and GINS integration into Mcm2-7-Cdc45 .

How do mutations in TopBP1 affect male fertility and meiotic processes?

Recent studies have identified a critical role for TopBP1 in male fertility through its function in meiotic sex chromosome inactivation (MSCI). A mouse line with mutations in the BRCT5 domain of TopBP1 (TopBP1 B5/B5) exhibits specific defects in male fertility:

Phenotypic Characterization:

• TopBP1 B5/B5 mice are viable but exhibit male-specific infertility

• Males show a threefold reduction in testis size and complete lack of spermatozoa

• Histological analysis reveals mainly spermatogonia and spermatocytes within the seminiferous epithelium

• Increased TUNEL-positive spermatocytes indicate elevated apoptosis

Molecular Mechanisms:

• TopBP1 B5/B5 spermatocytes progress through leptotene to pachytene stages but are unable to reach diplotene stage

• Unlike previous TopBP1 conditional knockouts, these mutants display largely normal:

  • Chromosome synapsis

  • Sex body formation

  • Recruitment of DNA damage response proteins to X and Y chromosomes

  • DNA repair during prophase I

• Single-cell RNA sequencing revealed that while MSCI is initiated, the dynamics of silencing progression and reinforcement are defective

• This defect is accompanied by abnormal localization of the RNA:DNA helicase Senataxin to chromatin loops of the XY chromosomes

Research Significance:
The TopBP1 B5/B5 mouse represents a valuable separation-of-function mutant that allows researchers to untangle XY silencing from sex body formation and DNA damage response recruitment, providing a unique model to study the establishment, maintenance, and progression of meiotic sex chromosome inactivation .

This research highlights an unexpected role for TopBP1 in meiotic processes distinct from its established functions in DNA replication and damage response.

What techniques can be used to study TopBP1 recruitment to chromatin and DNA damage sites?

Multiple complementary techniques can be employed to investigate TopBP1 recruitment to chromatin and DNA damage sites:

1. Chromatin Isolation from Xenopus Egg Extracts:

• Add sperm chromatin or DNA templates to egg extracts

• Isolate chromatin at different time points by centrifugation through a sucrose cushion

• Analyze bound proteins by Western blotting or mass spectrometry

• Can be combined with aphidicolin (DNA replication inhibitor) to accumulate replisomes on chromatin

2. DMAX System with Bead-Coupled DNA:

• Couple biotinylated DNA to magnetic streptavidin beads

• Incubate in Xenopus egg extracts

• Isolate beads, wash, and analyze bound proteins

• Enables identification of proteins recruited to DNA double-strand breaks

3. Immunodepletion and Add-Back:

• Deplete endogenous TopBP1 from extracts using antibodies

• Add back recombinant wild-type or mutant TopBP1

• Assess recruitment to chromatin and functional consequences

• Powerful for structure-function analysis of specific domains

4. CHROMASS Protocol:

• Combines chromatin isolation with mass spectrometry

• Allows comprehensive identification of proteins bound to chromatin

• Can be used to analyze changes in chromatin association following treatments or mutations

5. Microscopy-Based Approaches:

• Immunofluorescence of spread nuclei to visualize TopBP1 at specific nuclear structures

• Live-cell imaging with fluorescently tagged TopBP1 to track recruitment dynamics

• Super-resolution microscopy to determine precise localization patterns

Experimental Data from Recent Studies:
Research has shown that TopBP1 recruitment to DNA double-strand breaks requires phosphorylation-dependent interactions mediated by BRCT1, while BRCT7 contributes in a phosphorylation-independent manner . The BRCT5 domain functions downstream of recruitment to promote ATR-CHK1 signaling . For ultra-fine anaphase bridges (UFBs), TopBP1 recruitment occurs independently of BLM, MDC1, 53BP1, and PICH .

What are common challenges in expressing and purifying functional recombinant TopBP1?

Researchers face several technical challenges when working with recombinant TopBP1:

Expression Challenges:

• Size Limitations: Full-length TopBP1 (~180 kDa) often expresses poorly in bacterial systems

• Protein Folding: The multiple BRCT domains can cause folding issues during expression

• Post-translational Modifications: Bacterial systems lack machinery for critical phosphorylation events

Purification Obstacles:

• Protein Solubility: TopBP1 can form inclusion bodies, requiring optimization of solubilization conditions

• Proteolytic Degradation: The linker regions between BRCT domains are susceptible to proteolysis

• Maintaining Activity: Preserving the functional activity of purified protein can be difficult

Recommended Solutions:

For functional studies, many researchers have successfully used truncated versions of TopBP1 containing specific BRCT domains of interest rather than the full-length protein . This approach can circumvent many expression and purification challenges while still enabling mechanistic studies of domain-specific functions.

How can I detect and analyze TopBP1 interactions with partner proteins in Xenopus systems?

Multiple complementary approaches can be used to detect and characterize TopBP1 interactions in Xenopus systems:

Co-Immunoprecipitation (Co-IP) Methods:

• Extract Preparation:

  • Prepare nuclear or total extracts from Xenopus egg extracts

  • Add recombinant tagged TopBP1 or use antibodies against endogenous TopBP1

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

• Pulldown Strategies:

  • Use FLAG-tagged TopBP1 and anti-FLAG beads for clean pulldowns

  • For domain mapping, generate a series of internal deletion mutants to identify interaction regions

  • Salt titration can help determine interaction strength

• Analysis Methods:

  • Western blotting for known partners

  • Mass spectrometry for unbiased identification of interactors

  • Quantitative proteomics to compare wild-type vs. mutant interactions

Recent Applications and Findings:

• FLAG-tagged truncated TopBP1 (BRCT domains I-VI) has been used successfully to isolate TopBP1-associated proteins from lysates of replicating nuclei in Xenopus egg extracts

• This approach identified Treslin as a novel TopBP1-interacting protein involved in DNA replication initiation

• APEX2-tagged TopBP1 proximity biotinylation has been used to identify interactions with GINS complex subunits (Psf3, Psf2)

• For TopBP1-GINS interaction, both in vivo immunoprecipitation and in vitro binding assays with purified components have been employed to map binding surfaces

By combining these approaches, researchers can comprehensively characterize the TopBP1 interactome in Xenopus systems and map the domains responsible for specific protein-protein interactions.

What are the latest findings on TopBP1 function in DNA damage response pathways?

Recent studies have revealed new insights into TopBP1's role in DNA damage response:

TopBP1 in Ultra-Fine Anaphase Bridge Resolution:

• TopBP1 recruits TOP2A (Topoisomerase IIα) to ultra-fine anaphase bridges (UFBs) to aid in their resolution

• This recruitment occurs independently of established UFB factors like BLM, MDC1, 53BP1, and PICH

• TopBP1 depletion leads to an increase in BLM-positive UFBs, particularly at centromeres (C-UFBs)

• The BRCT5 domain is critical for TopBP1's association with UFBs

Structural Basis of ATR Activation:

• The DMAX system revealed that TopBP1 BRCT1 and BRCT7 are crucial for recruitment to DNA double-strand breaks

• BRCT1 mediates a phosphorylation-dependent interaction, while BRCT7 functions in a phosphorylation-independent manner

• BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1

• BRCT7 plays a dual role - one in recruitment and another, independent role in promoting ATR signaling

TopBP1 in DNA Replication:

• TopBP1 utilizes a bipartite binding mode to interact with the GINS complex

• This interaction involves both the GINI region and BRCT4 domain binding to opposite ends of the Psf1 A domain

• The interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon to GINS when part of the CMG complex

These findings collectively expand our understanding of TopBP1's multifaceted roles in maintaining genome stability through both its established function in ATR activation and newly discovered roles in resolving difficult-to-replicate DNA structures.

How is TopBP1 function regulated by post-translational modifications in Xenopus systems?

Post-translational modifications (PTMs) play crucial roles in regulating TopBP1 function, though this area remains incompletely characterized in Xenopus systems:

Phosphorylation:

• TopBP1 association with Treslin occurs in a Cdk2-dependent manner prior to DNA replication initiation in Xenopus egg extracts

• This phosphorylation-dependent interaction is critical for the loading of Cdc45 onto replication origins

• The interaction between TopBP1 BRCT1 and its binding partners at DNA double-strand breaks is phosphorylation-dependent, while BRCT7-mediated interactions are phosphorylation-independent

Research Methodologies for Studying TopBP1 PTMs:

• Mass Spectrometry-Based Approaches:

  • Isolation of TopBP1 from Xenopus egg extracts under different conditions

  • Phosphoproteomic analysis to identify modified residues

  • Quantitative comparison of modification states before and after DNA damage

• Phosphorylation-Specific Antibodies:

  • Development of antibodies recognizing specific phosphorylated residues

  • Western blotting to monitor phosphorylation status

• Phosphomimetic and Phosphodeficient Mutants:

  • Generation of S/T→E/D (phosphomimetic) and S/T→A (phosphodeficient) mutations

  • Functional testing in Xenopus egg extract systems

• Kinase Inhibitor Studies:

  • Treatment of extracts with specific kinase inhibitors (e.g., Cdk inhibitors)

  • Analysis of TopBP1 function and interactions

While the literature on Xenopus TopBP1 phosphorylation is not as extensive as for human TopBP1, the conservation of key regulatory mechanisms suggests that many phosphorylation events may be similar between species. Interestingly, while phosphorylation is clearly important for some TopBP1 functions, recombinant TopBP1 protein is sufficient for ATR activation, suggesting that regulation of some TopBP1 activities might not require post-translational modifications .

Future research should focus on comprehensive mapping of TopBP1 PTMs in Xenopus systems and determining their functional significance in various contexts such as normal replication, replication stress, and DNA damage.

What are the most promising future research directions for TopBP1 in Xenopus systems?

Several promising research directions emerge from current TopBP1 studies in Xenopus systems:

Structural Biology:

• Determining high-resolution structures of TopBP1 domains interacting with binding partners (e.g., ATR-ATRIP, GINS, Treslin)

• Analyzing conformational changes in TopBP1 upon binding to damaged DNA or during the replication cycle

• Investigating the structural basis for domain-specific functions in replication versus checkpoint control

Single-Molecule Studies:

• Applying single-molecule techniques to visualize TopBP1 recruitment and function at replication origins or DNA damage sites

• Real-time observation of the dynamics of TopBP1-mediated protein complex assembly

• Measuring the kinetics of TopBP1 association and dissociation at different DNA structures

Systems Biology:

• Comprehensive mapping of the TopBP1 interactome under different conditions (normal replication, replication stress, DNA damage)

• Integration of proteomic, genomic, and imaging data to build predictive models of TopBP1 function

• Network analysis to understand how TopBP1 coordinates multiple nuclear processes

Translational Research:

• Developing TopBP1-targeting approaches for potential therapeutic applications

• Investigating TopBP1 mutations associated with cancer or developmental disorders

• Using Xenopus systems as platforms for drug screening targeting the TopBP1-ATR pathway

The Xenopus system continues to offer unique advantages for studying TopBP1 function, including the ability to perform biochemical reconstitution, cell-free DNA replication, and checkpoint activation assays. The development of new technologies like DMAX enhances the power of this system for dissecting the molecular mechanisms of TopBP1 function in DNA metabolism and genome maintenance.

How do findings from Xenopus TopBP1 studies translate to human disease and therapeutic development?

Research on Xenopus TopBP1 provides valuable insights with translational implications for human health:

Cancer Biology Applications:

• TopBP1 is implicated in several human cancers, including breast cancer, lung cancer, and ovarian cancer

• The detailed mechanistic insights from Xenopus studies help explain how TopBP1 alterations contribute to genomic instability in cancer

• The DMAX system provides a platform for screening compounds that modulate TopBP1-ATR signaling, potentially identifying new cancer therapeutics

Developmental Biology Connections:

• TopBP1 knockout in mice causes embryonic lethality at the peri-implantation stage, indicating its essential role in early development

• TopBP1-deficient cells enter cellular senescence rather than apoptosis, with implications for aging and cancer

• The molecular mechanisms of TopBP1 function in DNA replication and repair elucidated in Xenopus likely apply to human embryonic development

Fertility Research:

• The TopBP1 B5/B5 mouse model revealed an unexpected role for TopBP1 in male fertility through meiotic sex chromosome inactivation

• This finding suggests potential links between TopBP1 mutations and human male infertility that warrant investigation

• Understanding the separation of TopBP1's functions in DNA damage response versus meiotic processes could lead to more targeted fertility treatments

Translational Challenges and Opportunities:

• Species Differences: While core functions are conserved, species-specific differences in TopBP1 regulation must be considered

• Therapeutic Targeting: The multi-functional nature of TopBP1 presents challenges for developing targeted therapeutics

• Diagnostic Potential: TopBP1 pathway defects could serve as biomarkers for certain cancers or fertility issues