RECQL5 Antibody

What is RECQL5 and why is it important to study?

RECQL5 belongs to the RecQ helicase family, which is crucial for maintaining genome stability. In mammals, there are five RecQ homologs (BLM, WRN, RECQL4, RECQL, and RECQL5), with defects in BLM, WRN, and RECQL4 being associated with cancer predisposition syndromes . RECQL5 functions as an important tumor suppressor, as demonstrated by studies showing that deletion of Recql5 in mice results in increased cancer susceptibility .

RECQL5's importance stems from its multifaceted roles in cellular processes:

• Regulates homologous recombination by displacing RAD51 from single-stranded DNA

• Acts as a general elongation factor during transcription

• Participates in DNA repair pathways, particularly in response to oxidative damage

• Protects cells during replication stress

• Suppresses genomic instability

These functions make RECQL5 a critical protein to study in the context of cancer biology, DNA repair mechanisms, and transcriptional regulation.

What are the structural and biochemical properties of RECQL5?

Human RECQL5 is characterized by:

• Length: 991 amino acid residues in the canonical form

• Molecular weight: 108.9 kDa (though observed molecular weights of 48 kDa and 130-140 kDa have been reported in Western blots)

• Subcellular localization: Nucleus

• Alternative splicing: Yields four different isoforms

Structurally, RECQL5 contains:

• A helicase domain with D1 and D2 subdomains that provide ATP-dependent DNA unwinding activity

• A RecQ C-terminal (RQC) domain

• An IRI module comprising the aN helix and KIX domain, which mediates interaction with RNA polymerase II

RECQL5 binds preferentially to splayed duplex, looped and single-stranded DNA structures, and likely unwinds DNA in a 3'-5' direction . Its helicase activity is ATP-dependent, requiring ATP hydrolysis particularly when displacing RAD51 from single-stranded DNA .

What are the optimal methodologies for detecting RECQL5 in Western blot experiments?

For successful Western blot detection of RECQL5, the following protocol is recommended:

Sample preparation:

• Harvest cells in an appropriate lysis buffer with protease inhibitors

• Enrich for nuclear proteins when possible (as RECQL5 is nuclear)

• Load 20-50 μg of total protein per lane

SDS-PAGE and transfer:

• Use 7-10% gels (suitable for the 108.9 kDa RECQL5 protein)

• Transfer to PVDF or nitrocellulose membrane using standard protocols

Immunoblotting conditions:

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Anti-RECQL5 at 1:1000-1:4000 dilution, overnight at 4°C

• Secondary antibody: Appropriate HRP-conjugated antibody at recommended dilution

• Detection: Standard ECL substrate

Expected results:

• Full-length RECQL5: ~108.9 kDa

• Alternative bands at 48 kDa and 130-140 kDa may be observed

• Verified positive controls include HEK-293T cells and mouse testis tissue

When troubleshooting low signal, consider concentrating the sample, increasing antibody concentration, extending incubation times, or using more sensitive detection methods.

How can immunofluorescence be optimized to visualize RECQL5 localization and interactions?

For optimal immunofluorescence detection of RECQL5:

Sample preparation:

• Grow cells on coverslips

• Fix with 4% paraformaldehyde (15 minutes, room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

Immunostaining protocol:

• Blocking: 5% BSA in PBS (1 hour, room temperature)

• Primary antibody: Anti-RECQL5 at 1:20-1:200 dilution (overnight, 4°C)

• Secondary antibody: Fluorophore-conjugated secondary antibody (1 hour, room temperature)

• Nuclear counterstain: DAPI

• Mount with anti-fade mounting medium

Co-localization studies:

• For DNA damage response: Co-stain with γH2AX (double-strand break marker)

• For homologous recombination: Co-stain with RAD51

• For BER/SSBR pathways: Co-stain with XRCC1

• For transcription studies: Co-stain with RNA polymerase II components

Imaging considerations:

• Use confocal microscopy for optimal resolution of nuclear structures

• For co-localization, capture sequential images to minimize bleed-through

• Employ appropriate metrics (Pearson's or Manders' coefficients) for quantification

HepG2 cells have been verified to show positive immunofluorescence staining with anti-RECQL5 antibodies .

How can RECQL5 antibodies be used to study its role in homologous recombination?

RECQL5 plays a critical role in regulating homologous recombination (HR) by displacing RAD51 from single-stranded DNA. RECQL5 antibodies can be used to investigate this function through several approaches:

Biochemical assays:

• Immunoprecipitation of RECQL5 followed by in vitro assays to study its ability to displace RAD51

• Western blot analysis to correlate RECQL5 expression with HR efficiency

• ChIP to detect RECQL5 binding to damaged DNA regions

Microscopy-based techniques:

• Immunofluorescence co-localization with HR factors (RAD51, BRCA1, BRCA2)

• BrdU incorporation assays to monitor HR-associated DNA synthesis

• Assessment of γH2AX foci resolution in relation to RECQL5 expression

Functional studies:

• Reporter assays distinguishing between short tract gene conversion (STGC) and long tract gene conversion (LTGC)

• Sister chromatid exchange (SCE) rate analysis in cells with altered RECQL5 levels

• Assessment of homologous recombination efficiency using DR-GFP or similar reporters

Research has demonstrated that RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. Through biochemical means and electron microscopy, it has been shown that RECQL5 displaces Rad51 from single-stranded DNA in a reaction requiring ATP hydrolysis and stimulated by RPA .

How can chromatin immunoprecipitation be optimized for RECQL5 despite technical challenges?

Chromatin immunoprecipitation (ChIP) with RECQL5 antibodies presents unique challenges as noted in the literature: "RECQL5 ChIP and ChIP-seq analysis was attempted, but sites of RECQL5 occupancy could not be reproducibly detected, possibly because RECQL5 is lowly expressed and represents a 'moving target' without appreciable DNA sequence preference" . Despite these challenges, researchers can employ several optimization strategies:

Enhanced cross-linking approach:

• Use dual cross-linking with formaldehyde plus protein-protein cross-linkers like DSG

• Increase cross-linking time (15-20 minutes instead of standard 10 minutes)

• Optimize sonication conditions for consistent chromatin fragmentation

Antibody optimization:

• Use increased antibody amounts (5-10 μg per IP)

• Test multiple antibodies targeting different RECQL5 epitopes

• Consider using tagged RECQL5 (HA, FLAG) in controlled expression systems

Specialized ChIP variations:

• ChIP-exo or ChIP-nexus for higher resolution

• Sequential ChIP (ChIP-reChIP) to identify regions where RECQL5 co-occupies with known interactors

• Employ cell synchronization to enrich for RECQL5 binding at specific cell cycle phases

Analytical considerations:

• Focus on regions where RECQL5 is likely to function (transcribed regions, DNA damage sites)

• Use more sensitive qPCR approaches with multiple primer sets

• Consider broader peak profiles when analyzing sequencing data

These optimizations may help overcome the challenges associated with RECQL5 ChIP experiments, though researchers should be prepared for extensive troubleshooting.

What experimental approaches can elucidate RECQL5's role in transcription regulation?

RECQL5's role as a transcription regulator can be investigated using several antibody-based approaches:

Genome-wide analyses:

• ChIP-seq for RNA polymerase II in RECQL5-depleted vs. control cells to assess RNAPII distribution

• Calculate traveling ratios (density of RNAPII in promoter-proximal region relative to gene body)

• Correlate RECQL5 expression with transcription elongation rates

Transcription elongation assays:

• DRB (5,6-dichlorobenzimidazole 1-beta-D ribofuranoside) release assays to measure elongation rates in vivo

• Time-resolved analysis of RNAPII arrival at different intron-exon junctions

• Assessment of nascent RNA synthesis using EU incorporation or GRO-seq

Protein interaction studies:

• Co-immunoprecipitation to confirm RECQL5 interaction with RNA polymerase II subunits

• Proximity ligation assay to visualize RECQL5-RNAPII interactions in situ

• Mass spectrometry analysis of RECQL5-associated transcription factors

Research has demonstrated that RECQL5 acts as a general elongation factor that decreases the elongation rate of RNAPII. RECQL5 depletion experiments revealed a genome-wide increase in RNAPII levels over transcription start sites with an inversion of relative RNAPII levels approximately 500 bp downstream. Conversely, RECQL5 overexpression had the opposite effect, reducing RNAPII density over promoters and increasing levels across transcribed regions .

How can researchers investigate RECQL5's role in DNA damage response using specific antibodies?

RECQL5 plays important roles in multiple DNA damage response pathways. To investigate these functions using antibodies:

Oxidative damage response:

• Combine RECQL5 expression analysis with measurement of 8-oxoguanine levels

• Assess RECQL5 recruitment to sites of oxidative damage using immunofluorescence

• Correlate RECQL5 levels with poly(ADP-ribosyl)ation status after oxidative stress

Replication stress response:

• BrdU incorporation assays to measure DNA replication in RECQL5-deficient vs. proficient cells after treatment with agents like camptothecin (CPT)

• Chromatin fractionation followed by Western blotting to assess RECQL5 recruitment during replication stress

• Immunofluorescence co-localization with replication markers (PCNA, RPA)

Double-strand break repair:

• Track γH2AX foci formation and resolution in relation to RECQL5 expression

• Co-localization studies with DSB repair factors

• Assess chromosomal aberrations in metaphase spreads from cells with altered RECQL5 levels

Functional assays:

• Clonogenic survival assays after treatment with different DNA-damaging agents

• Comet assay to assess DNA damage levels in RECQL5-manipulated cells

• Comparative genomic hybridization to detect genomic rearrangements

Research has shown that RECQL5-depleted cells accumulate endogenous DNA damage, are sensitive to oxidative stress, and show increased cellular poly(ADP-ribosyl)ation . Additionally, RECQL5 is particularly important for survival after camptothecin treatment, with RECQL5-deficient cells showing hypersensitivity to this topoisomerase I inhibitor .

How should researchers validate RECQL5 antibody specificity and address inconsistent results?

Rigorous validation of RECQL5 antibody specificity is essential for reliable experimental outcomes:

Comprehensive validation strategy:

Addressing inconsistent results:

• Compare experimental conditions (cell types, treatment protocols)

• Evaluate antibody batch variability

• Consider different RECQL5 isoforms or post-translational modifications

• Assess context-dependent protein interactions that may mask epitopes

• Test antibody performance across different applications (WB, IF, IP)

When working with RECQL5 antibodies, keep in mind that observed molecular weights of 48 kDa and 130-140 kDa have been reported in Western blots in addition to the expected 108.9 kDa band , potentially representing different isoforms or modified forms of the protein.

How can researchers reconcile seemingly contradictory data regarding RECQL5 function?

RECQL5 performs multiple cellular functions, which can sometimes lead to apparently contradictory observations. Researchers can employ several strategies to reconcile such data:

Methodological reconciliation:

• Carefully analyze methodological differences between studies

• Standardize experimental approaches and reagents

• Replicate contradictory findings under identical conditions

Biological context analysis:

• Consider cell type-specific effects

• Evaluate cell cycle dependence of observed phenomena

• Assess the influence of genetic background on RECQL5 function

Molecular mechanistic investigation:

• Determine if contradictions relate to different RECQL5 isoforms

• Examine post-translational modifications that might alter function

• Investigate context-dependent protein interactions

Integration of multiple approaches:

• Combine biochemical, cellular, and in vivo studies

• Use complementary techniques to assess the same function

• Develop comprehensive models accounting for RECQL5's multifaceted roles

An illustrative example comes from cancer studies: RECQL5 functions as a tumor suppressor (deletion leads to cancer susceptibility in mice) , yet it also promotes cancer cell survival under certain conditions (RECQL5 is important for camptothecin tolerance in colorectal cancer cells) . This apparent contradiction can be reconciled by understanding RECQL5's context-dependent functions in maintaining genome integrity, which can either prevent cancer initiation or help cancer cells survive stress conditions.

What novel approaches can be used to investigate RECQL5's role in cancer biology?

RECQL5's complex roles in cancer biology can be investigated through several innovative approaches:

Therapeutic response prediction:

• Immunohistochemical analysis of RECQL5 expression in patient tumor samples

• Correlation of RECQL5 levels with response to camptothecin-based therapies (irinotecan, topotecan)

• Development of RECQL5 as a potential biomarker for chemotherapy selection

Functional genomics approaches:

• CRISPR-Cas9 screens to identify synthetic lethal interactions with RECQL5 deficiency

• Domain-specific RECQL5 mutations to dissect function-specific cancer relationships

• Investigation of RECQL5 interplay with known oncogenes and tumor suppressors

Cancer model systems:

• Patient-derived xenografts with modulated RECQL5 expression

• Comparison of RECQL5 function across different cancer types

• In vivo imaging of RECQL5-dependent DNA repair in tumor models

Research has demonstrated that xenograft tumors derived from RECQL5-deficient HCT116 cells, but not those from the parental line, could be cured by a CPT-based therapy in nude mice, identifying RECQL5 as a major determinant for CPT resistance in colorectal cancer cells . This suggests that RECQL5 could serve as a biomarker for selecting patients who might benefit from irinotecan-based treatments for colon cancer.

How can researchers investigate the interplay between RECQL5 and other genome maintenance factors?

RECQL5 functions within a complex network of genome maintenance pathways. To understand its interplay with other factors:

Comparative analysis with other RecQ helicases:

• Study functional redundancy or specialization among RecQ family members

• Investigate synthetic phenotypes in cells with deficiencies in multiple RecQ helicases

• Compare biochemical activities using purified proteins

Investigation of pathway coordination:

• Examine how RECQL5 coordinates with ATRX in defining homologous recombination subpathways

• Study the relationship between RECQL5 and the Fanconi anemia pathway in interstrand crosslink repair

• Investigate RECQL5's interplay with mismatch repair factors

Systems biology approaches:

• Protein interaction network analysis centered on RECQL5

• Quantitative proteomics to identify damage-induced changes in RECQL5 complexes

• Computational modeling of genome maintenance pathway interactions

Genetic interaction studies:

• Double knockout/knockdown experiments to identify epistatic relationships

• Rescue experiments with domain-specific mutants

• CRISPR-based genetic screens to identify functional partners

Research has revealed distinct roles for ATRX and RECQL5 in homologous recombination, with ATRX being essential for extended DNA repair synthesis while RECQL5 influences long tract gene conversion and sister chromatid exchange formation . This exemplifies how RECQL5 functions in concert with other genome maintenance factors in defining specific DNA repair outcomes.