Recombinant Chicken RecQ-mediated genome instability protein 1 (RMI1), partial

What is RMI1 and what role does it play in DNA replication?

RMI1 (RecQ-mediated genome instability protein 1) is a crucial member of a conserved protein complex that includes BLM helicase and topoisomerase IIIα. This complex is essential for maintaining genomic stability during DNA replication. RMI1 specifically promotes normal replication fork progression and facilitates recovery from replication stress .

To study RMI1's role in replication, researchers commonly employ molecular combing techniques to analyze DNA replication at the single-molecule level. This methodology reveals that RMI1 depletion leads to impaired replication fork progression, which can be alleviated in cells lacking BLM. This indicates that RMI1 functions downstream of BLM in the replication elongation process .

For experimental investigation, researchers typically use RMI1 knockdown cell lines created through siRNA or shRNA approaches, followed by analysis of replication dynamics using DNA fiber analysis, BrdU incorporation studies, or molecular combing techniques.

How does RMI1 interact with other DNA maintenance proteins?

RMI1 forms direct protein-protein interactions with several key DNA maintenance factors. Most notably, RMI1 interacts with:

• BLM and topoisomerase IIIα to form the BTR (BLM-TopoIIIα-RMI1) complex

• FEN-1 (flap endonuclease 1) through its RQC domain and extreme C-terminal end

• RAD51, a key protein in homologous recombination repair

The interaction between RMI1 and FEN-1 is particularly interesting as it occurs independently of DNA, as demonstrated by experiments using benzonase or ethidium bromide treatment during binding assays . The RQC domain (amino acid residues 418-592) and C-terminus (amino acid residues 592-649) of RMI1 are responsible for this interaction, while the N-terminus and helicase domain do not bind FEN-1 .

To investigate these interactions, researchers commonly employ co-immunoprecipitation, GST pull-down assays, and yeast two-hybrid screening. Functional validation typically involves analyzing how these interactions affect enzymatic activities, such as RMI1's ability to stimulate FEN-1's endonucleolytic cleavage of 5'-flap DNA substrates.

What happens to cells when RMI1 is depleted?

RMI1 depletion results in several cellular phenotypes indicative of genome instability:

• Reduced cell viability

• Elevated levels of sister chromatid exchanges (SCEs), similar to Bloom syndrome cells

• Increased DNA damage accumulation

• Enhanced sensitivity to DNA damaging agents, particularly those that induce stalled or collapsed replication forks

• Impaired recovery from replication stress

• Accelerated tumorigenesis

• Increased G2/M cell cycle delay following genotoxic stress

Experimental methods to assess these phenotypes include cell survival assays (e.g., MTT or colony formation assays), sister chromatid exchange analysis, immunofluorescence staining for DNA damage markers (γH2AX), cell cycle analysis by flow cytometry, and chromosome spreads to detect structural abnormalities.

What is the molecular mechanism by which RMI1 promotes replication fork progression?

RMI1 ensures normal replication fork progression through several interconnected mechanisms:

• BLM-dependent function: RMI1 acts downstream of BLM to promote fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, suggesting that RMI1 counteracts a potentially deleterious effect of BLM at replication forks .

• Strand passage activity: The BLM-TopoIIIα-RMI1 complex exhibits strand passage activity in vitro, which is critical for resolving recombination intermediates that can arise during the processing of stalled replication forks .

• FEN-1 interaction: RMI1 stimulates FEN-1's endonucleolytic activity on 5'-flap DNA substrates, which is important for Okazaki fragment processing during lagging strand synthesis .

• Subnuclear localization: RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress, and this localization depends on interactions within the complex .

Methodologically, these mechanisms can be studied using:

• Single-molecule DNA fiber analysis to measure fork progression rates

• Biochemical assays to assess strand passage activity

• In vitro nuclease assays to measure FEN-1 stimulation

• Fluorescence microscopy to track subnuclear localization during replication stress

How does RMI1 contribute to telomere maintenance?

RMI1 plays significant roles in telomere maintenance through several mechanisms:

• Telomere localization: Both RMI1 and FEN-1 are constitutively present at telomeres, and their binding to telomeric chromatin is enhanced following DNA damage .

• FEN-1 recruitment: RMI1 is required for FEN-1 binding to telomeres in unperturbed cycling cells. Depletion of RMI1 reduces FEN-1 binding to telomeres, suggesting that RMI1 facilitates FEN-1 recruitment to telomeric regions .

• Protection from DNA damage: RECQ1-depleted cells show increased γH2AX-bound telomere sequence-specific DNA in response to hydroxyurea treatment, indicating that RMI1 protects telomeric sequences from DNA damage .

• Lagging strand synthesis: Given FEN-1's role in processing Okazaki fragments, RMI1's involvement in recruiting FEN-1 to telomeres suggests a specific function in lagging daughter telomere synthesis .

Research methodologies to investigate these functions include:

• Chromatin immunoprecipitation (ChIP) assays to measure protein binding to telomeric sequences

• γH2AX-ChIP to assess DNA damage at telomeres

• Telomere restriction fragment analysis to measure telomere length

• Fluorescence in situ hybridization (FISH) with telomere-specific probes

What is the role of RMI1 in homologous recombination DNA repair?

RMI1 plays a critical role in homologous recombination (HR) repair through multiple mechanisms:

• RAD51 interaction: Upon DNA damage, RMI1 forms nuclear foci at damaged regions, interacts with RAD51, and facilitates the recruitment of RAD51 to initiate homologous recombination .

• BTR complex: As part of the BLM-TopoIIIα-RMI1 complex, RMI1 helps resolve recombination intermediates, particularly double Holliday junctions, without crossing over .

• Suppression of illegitimate recombination: RMI1 functions with BLM and TopoIIIα to suppress inappropriate recombination events, which is reflected in the elevated sister chromatid exchange levels when RMI1 is depleted .

• Response to camptothecin: RMI1 depletion increases cell sensitivity to camptothecin (a topoisomerase I inhibitor), resulting in elevated levels of DNA double-strand breaks, stronger activation of the DNA damage response, and greater G2/M cell cycle delay .

Experimental approaches to study these functions include:

• Immunofluorescence microscopy to track RAD51 and RMI1 foci formation

• HR reporter assays to measure homologous recombination efficiency

• Sensitivity assays with DNA damaging agents like camptothecin

• Co-immunoprecipitation to detect protein-protein interactions during DNA damage response

How do post-translational modifications regulate RMI1 function?

While the search results do not directly address post-translational modifications (PTMs) of RMI1, this is an important research question given the regulation of DNA repair proteins by PTMs. Based on general knowledge of similar proteins:

Post-translational modifications likely regulate RMI1 function through:

• Phosphorylation: Potential phosphorylation by ATM, ATR, or other DNA damage response kinases may regulate RMI1's localization to damaged DNA or its interactions with partner proteins.

• SUMOylation/Ubiquitination: These modifications could regulate RMI1 stability or recruitment to specific nuclear compartments.

• Regulation of protein interactions: PTMs might modulate RMI1's interactions with BLM, TopoIIIα, FEN-1, or RAD51.

To investigate these regulatory mechanisms, researchers would typically employ:

• Mass spectrometry to identify specific modification sites

• Phospho-specific antibodies to track modifications during DNA damage response

• Mutational analysis of putative modification sites

• In vitro kinase assays to identify responsible enzymes

• Proteasome inhibitors to assess protein stability regulation

What are the species-specific differences in chicken RMI1 compared to human RMI1?

While the search results do not specifically address chicken RMI1, this comparative question is relevant for researchers working with recombinant chicken RMI1.

Species-specific differences between chicken and human RMI1 would likely include:

• Sequence conservation: Analysis of amino acid sequence conservation, particularly in functional domains like the RQC domain and C-terminal region that mediate protein interactions.

• Structural variations: Potential differences in tertiary structure that might affect protein-protein interactions or enzymatic activities.

• Interaction partners: While core interactions (BLM, TopoIIIα) are likely conserved, there may be species-specific interaction partners or regulatory mechanisms.

• Expression patterns: Potential differences in tissue-specific expression or developmental regulation.

Research approaches to investigate these differences include:

• Sequence alignment and phylogenetic analysis

• Structural modeling and prediction

• Comparative biochemical assays with recombinant proteins from both species

• Complementation experiments in knockout cell lines

What techniques are commonly used to study RMI1 function in DNA repair?

Several specialized techniques are employed to investigate RMI1's functions in DNA repair:

• Molecular combing: This single-molecule technique allows visualization and measurement of DNA replication dynamics, revealing how RMI1 affects replication fork progression .

• Chromatin immunoprecipitation (ChIP): Used to detect RMI1 binding to specific genomic regions, including telomeres, and to measure changes in binding following DNA damage .

• Immunofluorescence microscopy: Tracks the formation of RMI1 nuclear foci in response to DNA damage or replication stress, often co-stained with markers like γH2AX or RAD51 .

• Protein interaction assays: Co-immunoprecipitation, GST pull-down, and yeast two-hybrid approaches identify and characterize RMI1's interaction partners .

• In vitro biochemical assays: Assessing how RMI1 affects the enzymatic activities of partners like FEN-1, including flap endonuclease activity assays .

• Cell sensitivity assays: Determining how RMI1 depletion affects cellular response to DNA damaging agents, particularly those that induce replication stress .

These methodologies can be combined to build comprehensive understanding of RMI1's multifaceted roles in maintaining genome stability.

What are the best approaches for expressing and purifying recombinant RMI1?

Based on general practices for similar proteins, optimal approaches for expressing and purifying recombinant RMI1 include:

• Expression systems:

  • Bacterial expression (E. coli): Suitable for obtaining large quantities, though may lack post-translational modifications

  • Insect cell expression (baculovirus): Better for maintaining proper folding and modifications

  • Mammalian expression: Most likely to preserve native structure and modifications

• Tags and fusion proteins:

  • His-tag: For metal affinity purification

  • GST-tag: Enhances solubility and enables affinity purification

  • MBP-fusion: Improves solubility for difficult-to-express domains

• Purification strategy:

  • Initial affinity chromatography based on tag

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography as final polishing step

• Quality control:

  • SDS-PAGE and Western blotting to confirm purity and identity

  • Mass spectrometry for verification

  • Functional assays to confirm biological activity

For chicken RMI1 specifically, codon optimization for the expression system and careful consideration of potential species-specific post-translational modifications would be important factors.

How can researchers effectively study RMI1's role in replication stress response?

To effectively investigate RMI1's functions in replication stress response, researchers should consider these methodological approaches:

• Cell culture models:

  • RMI1 knockdown or knockout cell lines using RNAi or CRISPR-Cas9

  • Complementation with wild-type or mutant RMI1 to identify critical domains/residues

  • Treatment with replication stress-inducing agents (aphidicolin, hydroxyurea, camptothecin)

• Replication dynamics analysis:

  • DNA fiber assays to measure fork progression rates and restart efficiency

  • BrdU pulse-chase experiments to track newly synthesized DNA

  • Molecular combing for single-molecule analysis of replication structures

• Protein localization and interactions:

  • Live-cell imaging with fluorescently tagged RMI1 to track dynamics during stress

  • Proximity ligation assays to detect protein-protein interactions in situ

  • ChIP-seq to map genome-wide binding sites during normal and stressed conditions

• Functional readouts:

  • γH2AX foci formation to measure DNA damage accumulation

  • Cell cycle analysis to detect replication-associated checkpoints

  • Sister chromatid exchange assays to quantify recombination events

  • Cell survival assays following replication stress

These approaches can be integrated to develop a comprehensive understanding of how RMI1 contributes to genome stability during replication stress.

How might RMI1 dysfunction contribute to cancer development?

RMI1 dysfunction potentially contributes to cancer development through several mechanisms:

• Genomic instability: RMI1 inactivation causes elevated levels of sister chromatid exchange and general genomic instability, which are hallmarks of cancer cells .

• Accelerated tumorigenesis: Studies have shown that RMI1 deficiency can lead to accelerated tumorigenesis, highlighting its role as a tumor suppressor .

• Defective DNA repair: Impaired homologous recombination due to RMI1 dysfunction would increase the risk of mutations and chromosomal rearrangements .

• Telomere dysfunction: Given RMI1's role in telomere maintenance, its deficiency could lead to telomere instability, which is associated with cancer development .

• Replication stress sensitivity: Cancer cells often experience replication stress; RMI1 dysfunction would enhance the genomic instability resulting from this stress .

Research approaches to investigate these connections include:

• Analysis of RMI1 expression or mutation in cancer databases

• Mouse models with conditional RMI1 knockout to assess tumor development

• Assessment of synthetic lethality between RMI1 deficiency and other cancer-associated mutations

• Correlation of RMI1 status with cancer prognosis or therapy response

What are the potential therapeutic implications of targeting RMI1-dependent pathways?

Understanding RMI1 function has several potential therapeutic implications:

• Synthetic lethality: Identifying synthetic lethal interactions with RMI1 deficiency could reveal novel therapeutic targets for cancers with impaired RMI1 function.

• Sensitization to DNA damaging agents: Since RMI1 depletion increases sensitivity to camptothecin and other DNA damaging agents, RMI1 status might predict response to certain chemotherapies .

• Combination therapies: Targeting RMI1 or its interacting partners could potentially sensitize cancer cells to existing DNA-damaging treatments.

• Biomarker development: RMI1 expression or localization patterns could serve as biomarkers for genomic instability or treatment response.

Research strategies to explore these possibilities include:

• High-throughput screening for compounds that specifically target cells with RMI1 dysfunction

• Assessment of how RMI1 status affects response to various chemotherapeutics

• Development of small molecule inhibitors targeting RMI1-protein interactions

• Investigation of synthetic lethal interactions through CRISPR screens

How does RMI1 contribute to organism-level development and aging?

While the search results don't directly address developmental and aging roles, RMI1's fundamental functions suggest important contributions:

• Development: Given its role in maintaining genomic stability during DNA replication, RMI1 is likely essential for proper embryonic development when cells undergo rapid divisions.

• Stem cell maintenance: Genomic stability is crucial for stem cell function; RMI1 deficiency might affect stem cell pools throughout development and adult life.

• Aging processes: Genomic instability is a hallmark of aging; RMI1 dysfunction could accelerate age-related genomic deterioration and contribute to age-associated disorders.

• Telomere maintenance: RMI1's role in telomere stability connects it to cellular senescence and organismal aging .

Research approaches to investigate these broader implications include:

• Developmental studies in model organisms with RMI1 mutations

• Analysis of tissue-specific expression patterns throughout development and aging

• Investigation of RMI1 function in adult stem cell populations

• Longitudinal studies of genomic stability markers in relation to RMI1 status

How does RMI1 function compare to other RecQ-associated proteins?

RMI1 shares functional similarities and differences with other RecQ-associated proteins:

• Complex formation: Like WRN and BLM, RMI1 participates in multi-protein complexes crucial for DNA metabolism. The BLM-TopoIIIα-RMI1 complex is analogous to complexes formed by other RecQ helicases .

• FEN-1 interaction: Similar to WRN, BLM, RECQ5β, and RECQL4, RMI1 interacts with FEN-1, suggesting a conserved collaboration between RecQ family proteins and structure-specific nucleases .

• Domain structure: Unlike RecQ helicases, RMI1 itself lacks helicase activity but contains domains (like RQC) that are important for protein-protein interactions .

• Telomere functions: While several RecQ helicases function at telomeres, RMI1 has the specific role of facilitating FEN-1 recruitment to telomeric chromatin .

• DNA repair pathways: RMI1, like other RecQ-associated proteins, participates in homologous recombination repair, but with distinct roles in facilitating RAD51 recruitment .

Research methods to compare these functions include:

• Comparative biochemical assays

• Epistasis analysis in cells deficient for different RecQ-associated proteins

• Domain swap experiments to identify functional equivalences

• Comparative analysis of phenotypes in different knockout/knockdown models

What are the critical differences between full-length and partial RMI1 proteins?

While the search results don't specifically address partial versus full-length RMI1, this comparison is relevant for researchers working with recombinant protein forms:

Critical differences likely include:

• Functional domains: Partial RMI1 might lack certain functional domains, particularly affecting:

  • The RQC domain (residues 418-592) important for FEN-1 interaction

  • The C-terminal region (residues 592-649) also involved in protein interactions

  • N-terminal domains that might mediate other functions

• Protein interactions: Full-length RMI1 forms multiple protein interactions, while partial proteins might retain only subset of these capabilities:

  • BLM and TopoIIIα interactions forming the BTR complex

  • FEN-1 binding through RQC and C-terminal domains

  • RAD51 interactions important for homologous recombination

• Localization signals: Nuclear localization or subnuclear compartmentalization signals might be affected in partial proteins.

• Stimulatory activities: The ability to stimulate partner proteins (like FEN-1 endonuclease activity) might require full-length protein with native conformation .

Experimental approaches to investigate these differences include:

• Domain mapping through truncation analysis

• Complementation assays in RMI1-depleted cells

• Comparative biochemical activity assays

• Localization studies with tagged full-length versus partial proteins