Recombinant Arabidopsis thaliana DNA topoisomerase 1 (TOP1), partial

What is Arabidopsis thaliana DNA topoisomerase 1 (TOP1)?

DNA topoisomerase 1 (TOP1) is an essential enzyme that regulates DNA topology by alleviating tension during replication and transcription. In Arabidopsis thaliana, TOP1 plays critical roles in multiple cellular processes including DNA replication, repair, and recombination. The enzyme functions by breaking and changing the topological state of DNA molecules, which is crucial for cellular processes that involve DNA manipulation. TOP1 in Arabidopsis has been implicated in development, stem cell regulation, flowering time regulation, and adaptive responses to environmental stimuli . The protein contains several functional domains including a TOPRIM domain, an active center, and zinc-finger domains (ZFDs) at the C-terminus that are particularly important for targeting specific DNA structures .

How many isoforms of DNA topoisomerase 1 exist in Arabidopsis thaliana?

Arabidopsis thaliana contains two topoisomerase type I genes: TOP1α and TOP1β. These genes are tandemly located on chromosome 5 and share approximately 60% sequence similarity . Despite their structural similarities, they exhibit distinct biological functions. TOP1α plays predominant roles in plant development, as evidenced by the significant phenotypic effects observed in TOP1α knockout mutants. In contrast, downregulation of TOP1β has no detectable effects on plant development or physiology under standard growth conditions . This functional divergence suggests that TOP1α has evolved specialized roles in plant growth and development, while TOP1β may serve redundant or condition-specific functions that are not apparent under normal growth conditions.

What are the key structural domains of Arabidopsis thaliana TOP1 and their functions?

Arabidopsis thaliana TOP1α consists of several distinct functional domains:

• TOPRIM domain: Essential for catalytic activity, involved in DNA binding and metal ion coordination

• Active center: Contains the catalytic tyrosine residue (Y) that forms a transient covalent bond with DNA during the strand-breaking reaction

• Zinc-finger domains (ZFDs): Located at the C-terminus, with specific roles in targeting the enzyme to particular DNA structures

  • ZFD T1: Specifically required for targeting topoisomerase activity to Holliday junction (HJ)-like recombination intermediates

The importance of these domains has been demonstrated through complementation studies. Mutants lacking the TOPRIM domain or carrying mutations in the catalytic tyrosine of the active center display embryo lethality when expressed in a TOP1α knockout background . Interestingly, simultaneous removal of the ZFDs can overcome this lethality, suggesting complex interactions between the catalytic and targeting functions of the enzyme.

What phenotypes are observed in TOP1α mutants in Arabidopsis?

TOP1α mutants in Arabidopsis display a spectrum of phenotypes depending on the nature of the mutation:

• Complete knockout mutants (CRISPR/Cas-induced):

  • Viable but exhibit growth retardation

  • Meiotic defects affecting fertility

  • Can be complemented by expression of the complete protein

• fas5 mutant (specific allele of TOP1α):

  • Constitutive shade avoidance syndrome

  • Leaf hyponasty and petiole elongation

  • Lighter leaf color

  • Early bolting/flowering

• Domain-specific effects:

  • Expression of TOP1α missing the TOPRIM domain or with mutations in the catalytic tyrosine leads to embryo lethality

  • TOP1α without ZFDs shows partial complementation of growth defects in specific genetic backgrounds

These varied phenotypes highlight the multifaceted roles of TOP1α in plant development, from basic cellular functions to complex developmental processes and environmental responses.

What expression systems are most effective for recombinant Arabidopsis TOP1?

Based on available research, several expression systems have been successfully used for producing recombinant Arabidopsis TOP1:

• Escherichia coli expression system: Most commonly used for biochemical and structural studies. For example, researchers have successfully expressed portions of TOP1α lacking the N-terminal signal peptide (ΔSPTOP1) in E. coli for crystallization studies . This system allows for high yield but may require optimization for proper folding of plant proteins.

• In vitro transcription/translation systems: Useful for producing active enzyme for biochemical assays without extensive purification steps. This approach can help avoid issues with toxicity that might occur when expressing topoisomerases in bacterial systems.

When expressing TOP1 in bacterial systems, several considerations should be addressed:

• Temperature optimization (typically lowered to 16-18°C after induction)

• Use of specialized E. coli strains that supply rare codons

• Co-expression with chaperones to improve folding

• Addition of appropriate fusion tags (His, GST, or MBP) to enhance solubility and facilitate purification

The choice of expression system should be guided by the specific research question and the requirements for protein quantity, purity, and activity.

What are the critical factors for maintaining enzymatic activity during purification?

Maintaining the enzymatic activity of recombinant TOP1 during purification requires careful attention to several factors:

• Redox conditions: Arabidopsis TOP1 contains multiple cysteine residues that can form disulfide bonds. The TOP1 structure reveals conserved cysteines (C548/460, C611/523, and C699/611 in TOP1/TOP2) in the peptidase domain . Maintaining appropriate reducing conditions (typically with DTT or β-mercaptoethanol) is essential to preserve the native conformation and activity.

• Buffer composition:

  • pH maintenance (typically pH 7.5-8.0)

  • Inclusion of glycerol (10-20%) to stabilize the protein

  • Addition of protease inhibitors to prevent degradation

  • Presence of Mg²⁺ ions, which are necessary for catalytic activity

• Temperature control: Purification should be performed at 4°C to minimize protein denaturation and proteolytic degradation.

• Rapid processing: Minimize the time between cell lysis and final purification step to reduce exposure to proteases and oxidizing conditions.

• Storage conditions: Purified TOP1 should be stored in small aliquots at -80°C with glycerol to prevent freeze-thaw damage and maintain long-term stability.

Following these guidelines helps preserve the structural integrity and enzymatic activity of recombinant TOP1 throughout the purification process.

How can the specific role of the zinc-finger domain T1 be studied experimentally?

The zinc-finger domain T1 (ZFD T1) has been identified as critical for targeting topoisomerase activity to Holliday junction (HJ)-like recombination intermediates . To study this domain's function experimentally, several approaches can be employed:

• Domain deletion and mutation analysis:

  • Generate constructs with specific deletions or point mutations in ZFD T1

  • Express these constructs in TOP1α knockout backgrounds

  • Assess complementation of phenotypes (growth, meiosis, DNA repair)

• Structure-guided mutagenesis:

  • Identify conserved residues within ZFD T1 through sequence alignment

  • Create point mutations that disrupt zinc coordination or DNA binding

  • Evaluate the effects on protein function in vivo and in vitro

• Direct binding assays:

  • Use electrophoretic mobility shift assays (EMSAs) with purified WT and mutant proteins

  • Test binding to various DNA structures (HJs, D-loops, replication forks)

  • Quantify binding affinities and specificities

• Functional rescue experiments:

  • Express bacterial resolvases like RusA, which process HJ intermediates, in TOP1α mutant backgrounds

  • Assess whether these enzymes can rescue specific phenotypes associated with ZFD T1 dysfunction

A particularly informative approach used in published research involved expressing the E. coli resolvase RusA in a background where TOP1α lacked ZFDs. This rescued root growth defects, confirming that ZFD T1 specifically targets HJ-like recombination intermediates .

What methodologies are effective for studying TOP1α's role in meiotic recombination?

Investigating TOP1α's role in meiotic recombination requires a combination of cytological, genetic, and molecular approaches:

• Cytological analysis of meiotic progression:

  • Chromosome spreading techniques to visualize meiotic stages

  • Immunolocalization of meiotic proteins (recombination enzymes, synaptonemal complex components)

  • Fluorescence in situ hybridization (FISH) to track specific chromosomal regions

• Genetic analysis of recombination rates:

  • Tetrad analysis using fluorescent markers

  • Measurement of crossover frequencies between linked markers

  • Analysis of non-Mendelian segregation patterns

• Physical detection of recombination intermediates:

  • Two-dimensional gel electrophoresis to detect Holliday junctions

  • Chromatin immunoprecipitation (ChIP) to identify TOP1α binding sites

  • Pulsed-field gel electrophoresis to analyze chromosome integrity

• Interaction studies with known meiotic factors:

  • Yeast two-hybrid or co-immunoprecipitation to identify protein partners

  • Genetic interaction studies with mutations in genes like ATM, which functions in DNA repair pathways

Research has shown that TOP1α plays a role in promoting homologous chromosome crossover formation and may function in shared DNA repair pathways with ATM in Arabidopsis microspore mother cells . The absence of TOP1α leads to aberrant chromatin behavior during meiotic division, highlighting its importance in ensuring proper chromosome segregation.

How does TOP1α interact with the ATM-mediated DNA repair pathway?

The interaction between TOP1α and the ATM-mediated DNA repair pathway represents an important area of research with implications for understanding genome stability mechanisms. Based on current research:

• Functional relationship:

  • TOP1α potentially functions in a shared DNA repair pathway with ATM (ATAXIA TELANGIECTASIA MUTATED) in Arabidopsis

  • ATM acts as a critical regulator in the DNA damage response pathway, promoting repair through phosphorylation of key proteins involved in cell cycle arrest checkpoints

• Mechanistic interactions:

  • ATM responds to DNA double-strand breaks (DSBs), which can arise during TOP1 activity if the enzyme's catalytic cycle is disrupted

  • TOP1α activity may generate DNA structures or intermediates that require ATM signaling for proper processing

  • Both proteins influence meiotic processes, with ATM playing roles in DSB formation/processing, DNA repair, crossover formation, and synaptonemal complex assembly

• Experimental approaches to study this interaction:

  • Generate and characterize top1α atm double mutants to assess genetic interactions

  • Analyze phosphoproteome changes in top1α mutants to identify ATM-dependent phosphorylation events

  • Examine localization patterns of DNA repair proteins in top1α mutants during meiosis

  • Assess TOP1α recruitment to DSB sites in wild-type vs. atm mutant backgrounds

Understanding this interaction provides insight into how topological changes in DNA are coordinated with damage sensing and repair pathways to maintain genome integrity during both mitotic and meiotic cell cycles.

How does TOP1α regulate flowering time and shade avoidance response?

Recent research has uncovered unexpected roles for TOP1α in flowering time regulation and shade avoidance response. The molecular mechanisms underlying these phenotypes involve complex signaling networks:

• Shade avoidance response regulation:

  • The fas5 mutant (an allele of TOP1α) exhibits constitutive shade avoidance syndrome, characterized by leaf hyponasty, petiole elongation, lighter leaf color, and early bolting

  • RNA sequencing confirmed activation of shade avoidance gene pathways in fas5 mutant plants even under normal light conditions

  • This suggests TOP1α normally suppresses shade avoidance responses in the absence of actual shade stimulus

• Flowering time regulation:

  • TOP1α mutations lead to early bolting/flowering

  • RNA sequencing revealed repression of many genes controlling floral meristem identity and organ morphogenesis in fas5 mutants

  • This indicates that TOP1α is required for proper expression of flowering-related genes

• Potential molecular mechanisms:

  • TOP1α may affect chromatin structure at loci involved in light signaling and flowering

  • Changes in DNA topology could influence transcription factor accessibility to promoters

  • Altered DNA supercoiling may affect long-range chromosomal interactions important for gene expression coordination

The dual role of TOP1α in both developmental timing and environmental response pathways highlights how fundamental DNA topology management can influence complex plant adaptive behaviors. This represents an emerging area where basic DNA metabolism intersects with higher-order plant physiology.

What are the relationships between different TOP1α domains and their specific biological functions?

The multi-domain structure of TOP1α contributes to its diverse biological functions through specialized activities:

Interestingly, complex interactions exist between these domains:

• Mutations in the TOPRIM domain or active center cause embryo lethality

• This lethality can be overcome by simultaneous removal of the ZFDs

• This suggests that the targeting function of ZFDs, when coupled with an inactive enzyme, creates toxic intermediates that prevent proper DNA processing

Additionally, when combined with mutations in the nuclease MUS81, TOP1α knockout is embryo lethal. Expression of TOP1α without ZFDs provides only partial complementation in this background, while the complete protein restores root length to mus81-1 mutant levels . This indicates that ZFD T1 is specifically required for processing DNA structures that would otherwise be resolved by MUS81.

These findings reveal that TOP1α domains work in concert to balance DNA topology management with specific targeting to DNA structures requiring topoisomerase activity.

How can genome-wide studies advance our understanding of TOP1α function?

Genome-wide approaches offer powerful tools to expand our understanding of TOP1α's diverse functions in Arabidopsis:

• Chromatin immunoprecipitation sequencing (ChIP-seq):

  • Identify genome-wide binding sites of TOP1α

  • Correlate binding patterns with gene expression, chromatin accessibility, and DNA replication

  • Compare binding profiles under different developmental stages or environmental conditions

• RNA sequencing (RNA-seq):

  • Analyze transcriptome changes in top1α mutants across tissues and conditions

  • As demonstrated in fas5 mutants, RNA-seq revealed activation of shade avoidance pathways and repression of floral identity genes

  • Identify direct vs. indirect effects through integration with ChIP-seq data

• Genome-wide association studies (GWAS):

  • Identify natural variation in TOP1α and associated phenotypes

  • Discover genetic modifiers that interact with TOP1α function

• Chromosome conformation capture techniques (Hi-C):

  • Investigate how TOP1α affects three-dimensional genome organization

  • Explore connections between DNA topology and higher-order chromatin structure

• DNA breakage and repair mapping:

  • END-seq or BLESS to map DNA breaks genome-wide in wild-type vs. top1α mutants

  • Identify genomic regions particularly dependent on TOP1α activity

Integration of these approaches can provide a comprehensive understanding of how TOP1α functions at the intersection of DNA topology, gene expression, DNA repair, and developmental regulation. For example, combined analysis of chromatin structure and transcriptome data from fas5 mutants could reveal how changes in DNA topology influence the shade avoidance and flowering pathways at the molecular level.

What are the most common challenges when working with recombinant TOP1 and how can they be addressed?

Researchers working with recombinant TOP1 frequently encounter several challenges:

• Low expression levels or inclusion body formation:

  • Solution: Optimize expression conditions (reduce temperature to 16-18°C, use specialized E. coli strains)

  • Try fusion tags that enhance solubility (MBP tag often works better than His or GST for topoisomerases)

  • Consider expressing individual domains separately for specific analyses

• Loss of enzymatic activity during purification:

  • Solution: Maintain reducing conditions throughout purification

  • Include glycerol (10-20%) in all buffers to stabilize protein structure

  • Minimize time between cell lysis and final purification step

• Protein instability during storage:

  • Solution: Store in small aliquots at -80°C with 25-50% glycerol

  • Avoid repeated freeze-thaw cycles

  • Add stabilizers like trehalose or specific DNA oligonucleotides

• Contaminating nuclease activity:

  • Solution: Include EDTA in final storage buffer (but remove before activity assays)

  • Use high-salt washes during purification to remove DNA-binding contaminants

  • Test preparations with supercoiled plasmid DNA to check for non-specific degradation

• Difficulty distinguishing TOP1α from TOP1β activities:

  • Solution: Design isoform-specific antibodies targeting unique regions

  • Express recombinant proteins with distinguishable tags

  • Use genetic backgrounds lacking one isoform for in vivo studies

Addressing these challenges requires careful optimization of protocols for the specific research question and adapting published methods to the particular requirements of Arabidopsis TOP1.

What controls should be included when studying TOP1 mutants?

Proper experimental design for studying TOP1 mutants requires several types of controls:

• Genetic controls:

  • Wild-type plants (same ecotype as mutant background)

  • Heterozygous mutants (to assess dose-dependent effects)

  • Complemented lines expressing the wild-type gene

  • Domain-specific complementation (e.g., constructs lacking specific domains like ZFDs)

• Phenotypic assessment controls:

  • Growth under different light conditions (considering TOP1α's role in shade avoidance)

  • Stress treatments to assess DNA damage response (oxidative stress, DNA damaging agents)

  • Analysis across developmental stages (particularly during reproductive development)

• Molecular controls:

  • qRT-PCR to confirm absence of TOP1α transcript in knockout lines

  • Western blotting to verify protein expression levels in complementation lines

  • Activity assays to confirm enzymatic function of complementing constructs

• Specialized controls for specific experiments:

  • For meiotic studies: Include known meiotic mutants (e.g., atm) as comparative controls

  • For growth studies: Include mus81 mutants when studying ZFD functions

  • For gene expression studies: Include controls for specific pathways being examined (e.g., shade response mutants)

Research has demonstrated the value of specialized controls, such as expressing the E. coli resolvase RusA to test whether Holliday junction processing is specifically affected in ZFD T1 mutants . This type of functional complementation provides mechanistic insight beyond simple phenotypic rescue.

How can researchers distinguish between direct and indirect effects of TOP1α mutation?

Distinguishing direct from indirect effects of TOP1α mutation is crucial for understanding its true biological functions. Several approaches can address this challenge:

• Temporal analysis of molecular changes:

  • Use inducible systems (e.g., estrogen-inducible TOP1α RNAi) to track immediate vs. delayed responses

  • Time-course experiments following TOP1α inhibition or inactivation

  • Early changes are more likely to represent direct effects of TOP1α loss

• Domain-specific mutations:

  • Compare phenotypes between catalytically inactive vs. targeting-deficient mutants

  • For example, studying ZFD deletions separately from active site mutations

  • Different functional domains may mediate distinct cellular processes

• Integration of multiple data types:

  • Combine ChIP-seq data (TOP1α binding sites) with RNA-seq (expression changes)

  • Genes showing both TOP1α binding and expression changes are likely direct targets

  • Changes in genes without TOP1α binding suggest indirect effects

• Genetic interaction studies:

  • Analyze double mutants between top1α and genes in suspected pathways

  • Epistasis analysis can reveal pathway relationships

  • For example, top1α mus81 double mutants show embryo lethality, suggesting distinct but convergent functions

• Acute vs. chronic effects:

  • Compare effects of chemical inhibition (acute) with genetic knockout (chronic)

  • Developmental compensation may mask some functions in stable mutant lines

  • Conditional systems can reveal functions obscured by developmental adaptation

These approaches can help dissect the complex network of effects following TOP1α disruption and identify the primary molecular functions of this multifaceted enzyme in plant biology.

What are the emerging areas of TOP1α research in plant biology?

Several promising research directions are emerging in the field of plant TOP1α biology:

• Stress response regulation:

  • Investigating TOP1α's role in adaptation to abiotic stresses beyond shade avoidance

  • Exploring potential functions in biotic stress responses and plant immunity

  • Understanding how TOP1α activity is modulated under stress conditions

• Epigenetic regulation:

  • Examining connections between DNA topology and epigenetic modifications

  • Investigating how TOP1α affects DNA methylation patterns and histone modifications

  • Understanding TOP1α's potential role in transgenerational stress memory

• Organellar functions:

  • Detailed exploration of TOP1α's role in chloroplast and mitochondrial genome maintenance

  • Investigating coordination between nuclear, chloroplast, and mitochondrial genomes

  • Understanding how organellar TOP1α activity influences plant energy metabolism

• Evolutionary perspectives:

  • Comparative analysis of TOP1α across plant species with different genome sizes and complexities

  • Investigation of TOP1α specialization in different plant lineages

  • Understanding why plants maintain both TOP1α and TOP1β despite functional divergence

• Biotechnological applications:

  • Developing TOP1α-based tools for plant genome engineering

  • Exploring TOP1α manipulation to enhance stress tolerance or developmental traits

  • Using knowledge of TOP1α function to improve plant transformation efficiency

The continuing investigation of TOP1α's multifaceted roles will likely reveal new insights into fundamental aspects of plant genome maintenance, development, and environmental adaptation.

What technologies might advance our understanding of TOP1 function?

Emerging technologies offer exciting possibilities for deeper insights into TOP1 function:

• Single-cell omics approaches:

  • Single-cell RNA-seq to capture cell-type specific responses to TOP1α disruption

  • Single-cell ATAC-seq to examine changes in chromatin accessibility

  • Spatial transcriptomics to map TOP1α-dependent expression patterns in intact tissues

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize TOP1α localization at specific chromatin structures

  • Live-cell imaging with fluorescently tagged TOP1α to track dynamics during DNA replication and repair

  • Correlative light and electron microscopy to connect TOP1α activity with ultrastructural changes

• Novel biochemical approaches:

  • CRISPR-based recruitment of TOP1α to specific genomic loci

  • In vivo topological state mapping using techniques like TwistSeq

  • Protein engineering to create TOP1α variants with altered substrate specificity or regulation

• Computational modeling:

  • Molecular dynamics simulations of TOP1α interaction with different DNA structures

  • Systems biology approaches to model TOP1α's role in genetic and epigenetic networks

  • Prediction of DNA regions likely to require TOP1α activity based on sequence and structural features

Integration of these technologies with traditional genetic and biochemical approaches will provide unprecedented insights into how TOP1α coordinates DNA topology changes with fundamental cellular processes and complex developmental programs in plants.