HLTF Antibody

What is HLTF and what are its key functions in cellular processes?

HLTF is a multifunctional protein that serves primarily as a DNA translocase involved in post-replication DNA repair. HLTF has several critical functions:

• Fork reversal activity: HLTF promotes replication fork remodeling during replication stress, preventing other mechanisms of replication stress tolerance in cancer cells . EM studies have confirmed HLTF as a bona fide fork reversal protein in human cells, with HLTF-KO cell lines exhibiting a significant 2-3 fold reduction in reversed fork frequency .

• Ubiquitin ligase activity: Similar to yeast Rad5, HLTF functions as a ubiquitin ligase that promotes the polyubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue . This activity is crucial for error-free post-replication repair of damaged DNA.

• Interaction with DNA repair proteins: HLTF physically interacts with multiple DNA repair proteins including the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes . It also interacts with mismatch repair (MMR) proteins, with human HLTF shown to directly interact with MSH2 .

What is the molecular weight of HLTF and how should it appear on Western blots?

HLTF has a molecular weight of approximately 130 kDa on SDS-PAGE gels . When working with HLTF antibodies:

• The immunoprecipitated HLTF typically runs at the predicted molecular weight of 116 kDa

• Non-immunoprecipitated HLTF in input lanes may run at a slightly higher molecular weight and show several additional bands

• This pattern is consistent with manufacturer specifications for commercial HLTF antibodies

In which tissues and cell types is HLTF expressed?

HLTF is expressed in various cell types including:

• Cancer cell lines such as HCT116 and HeLa cells

• Primary human macrophages (MDMs), with expression detected at day 4 or day 7 following differentiation of freshly isolated monocytes

• Multiple tissues in human and animal models, though expression levels vary significantly

Interestingly, HLTF expression in non-dividing cells like macrophages suggests potential functions beyond its established role in S-phase DNA repair .

How can researchers detect altered HLTF function in replication stress models?

To detect HLTF's role in replication stress, several experimental approaches are recommended:

DNA fiber analysis to assess replication dynamics:

• Pulse-label cells with IdU followed by CldU in the presence of replication stressors (e.g., 50μM HU or MMC)

• In wild-type cells, replication tracts shorten by ~30% upon drug treatment

• HLTF-KO cells exhibit unrestrained fork progression (no shortening of tracts)

• This phenotype can be observed in various cell types including cancer cell lines (K562) and non-cancerous cells (RPE1)

Detection of fork reversal by electron microscopy:

• Isolate replication intermediates after exposing cells to replication stressors

• Use in vivo psoralen crosslinking and EM analysis

• Quantify reversed fork structures (typically ~23% of replication intermediates in wild-type cells treated with HU)

• HLTF-KO cells show 2-3 fold reduction in reversed fork frequency

S1 nuclease assay for discontinuous replication:

• Treat cells with replication stressors (e.g., 50μM HU)

• Incubate permeabilized cells with/without S1 nuclease

• S1 treatment specifically shortens replication tracts in HLTF-KO cells under stress conditions, indicating discontinuous replication with ssDNA gaps

What controls should be included when studying HLTF's role in DNA repair mechanisms?

Comprehensive controls are essential when investigating HLTF function:

Genetic controls:

• HLTF-KO cell lines (multiple clones recommended to rule out off-target effects)

• HLTF-HIRAN domain mutants (to distinguish effects of different HLTF domains)

• Complementation with wild-type or mutant HLTF expression constructs

• Double knockout models (e.g., HLTF-PRIMPOL-dKO) to study pathway dependencies

Experimental controls for immunoprecipitation:

• DNase treatment controls to distinguish DNA-mediated from direct protein-protein interactions

• Empty vector controls for tagged-protein expression systems

• Negative control antibodies for immunoprecipitation

• Input samples (5-10% of lysate) to verify protein expression levels

Technical validation:

• siRNA-resistant HLTF constructs for rescue experiments

• Multiple HLTF antibodies targeting different epitopes

• Cell cycle synchronization to account for cell cycle-dependent effects

How can researchers distinguish between HLTF's functions and those of its related proteins SHPRH and SMARCAL1?

HLTF belongs to a small family of DNA translocases including SMARCAL1 and ZRANB3 that catalyze fork regression activity. To distinguish their functions:

Protein-specific interactions:

• HLTF selectively interacts with MSH2 but not MLH1

• SHPRH interacts with MLH1 but not MSH2

• These differential interactions persist after DNase treatment, confirming specific protein-protein interactions

Functional assays:

• MNNG sensitivity: SHPRH-KO cells show moderate resistance to alkylating agents, whereas HLTF-KO cells remain sensitive

• Double knockout (HLTF/SHPRH-dKO) shows similar resistance to SHPRH-KO alone, suggesting non-redundant functions

Domain-specific assays:

• The HIRAN domain of HLTF is required for restraining replication fork progression

• HLTF's RING domain mediates its ubiquitin ligase activity

• Domain-specific mutations can help differentiate the multiple functions of HLTF

What are the methodological considerations for studying HLTF in cancer models?

When investigating HLTF in cancer contexts, researchers should consider:

Expression analysis:

Experimental models:

• Cell line-derived xenograft (CDX) models with controlled HLTF status are valuable

• For colorectal cancer studies, HCT116-derived models with shRNA knockdown of HLTF have been established

• Both the tumor cells and tumor microenvironment (TME) should be considered, as HLTF status in both compartments affects tumor biology

Response to therapy:

• HLTF-deficient cells show reduced DNA damage signaling (ATR/ATM) after replication stress

• HLTF loss can affect sensitivity to chemotherapeutic agents

• Combined analysis of HLTF with other DNA repair factors provides more comprehensive insights into therapy response

Why might HLTF antibodies show inconsistent banding patterns in Western blot applications?

Several factors can contribute to variable HLTF detection patterns:

Post-translational modifications:

• HLTF undergoes various modifications that can alter its migration pattern

• Ubiquitination of HLTF (particularly in HIV-1 Vpr-expressing cells) can generate additional bands

• Phosphorylation status may vary depending on cellular context

Protein degradation mechanisms:

• HLTF can be targeted for proteasomal degradation by viral proteins like HIV-1 Vpr

• Endogenous degradation pathways may generate cleavage products

• Use of protease inhibitors during sample preparation is critical

Technical recommendations:

• Freshly prepared samples typically yield cleaner results

• Include positive controls from cells known to express HLTF

• Compare results using antibodies targeting different HLTF epitopes

• Verify specificity using HLTF-knockout lysates as negative controls

How can researchers optimize immunoprecipitation protocols for studying HLTF interactions?

For successful HLTF co-immunoprecipitation studies:

Sample preparation:

• Nuclear fractionation is recommended as HLTF primarily localizes to the nucleus

• Use of 1:50 dilution of HLTF antibody has been validated for immunoprecipitation

• Include DNase treatment controls to distinguish DNA-mediated from direct protein interactions

Buffer conditions:

• Salt concentration is critical: too high may disrupt interactions, too low increases nonspecific binding

• Mild detergents (0.1% NP-40) help maintain protein complex integrity

• Consider crosslinking approaches for transient interactions

Validation approaches:

• Reciprocal IP (pull down with antibody against interaction partner)

• Both endogenous and tagged-protein approaches provide complementary evidence

• Follow interactions under different conditions (e.g., DNA damage, cell cycle phases)

What pitfalls should researchers avoid when analyzing HLTF function in cell cycle studies?

When studying HLTF's impact on cell cycle progression:

Common pitfalls:

• Cell type-specific effects: HLTF loss impacts different cell types differently

• Failure to account for cell synchronization: HLTF's effects are most pronounced during S-phase

• Incomplete pathway analysis: HLTF functions within complex DNA repair networks

Recommended approaches:

• Quantitative image-based cytometry (QIBC) for cell cycle analysis provides superior resolution compared to standard flow cytometry

• Dual pulse labeling (e.g., EdU/BrdU) allows tracking of cell progression through S-phase

• Combined analysis of DNA content and replication markers provides more complete cell cycle assessment

Key considerations:

• HLTF-KO cells progress faster through S-phase under replication stress conditions

• This accelerated progression depends partly on the primase PRIMPOL

• Cell cycle differences emerge after replication stress induction, not under normal conditions

How do HLTF mutations affect its function in different experimental systems?

HLTF contains several functional domains with distinct roles:

HIRAN domain mutations:

ATPase domain mutations:

• The DE557,558AA ATPase mutant lacks translocase activity but retains protein interaction capabilities

• This mutation abolishes fork regression activity but not protein complex formation

• Useful for separating enzymatic from scaffolding functions of HLTF

RING domain mutations:

• Affect HLTF's ubiquitin ligase activity

• Can be used to study PCNA polyubiquitination independently of fork remodeling

• Important for understanding pathway-specific functions

What are the best experimental approaches for studying HLTF's role at the replication fork?

To effectively study HLTF's functions at replication forks:

In vitro fork regression assays:

• Purified HLTF protein can process model replication fork structures

• HLTF concertedly unwinds and anneals the nascent and parental strands without exposing extended single-stranded regions

• SSB proteins do not affect HLTF-mediated fork regression, unlike conventional helicase assays

Chromatin association studies:

• iPOND (isolation of Proteins On Nascent DNA) to identify HLTF recruitment to replication forks

• SIRF (in situ analysis of protein interactions at DNA replication forks) for visualization of HLTF at individual forks

• ChIP-seq approaches for genome-wide analysis of HLTF binding sites

Protein-protein interaction mapping:

• HLTF interacts with key replication and repair factors including PCNA, Rad18, Mms2, and Ubc13

• These interactions occur constitutively and are not significantly affected by DNA damage induction

• Both endogenous and tagged protein approaches provide complementary information

How should researchers interpret contradictory findings on HLTF's role in cancer progression?

The literature contains seemingly contradictory findings about HLTF in cancer:

Tumor suppressor evidence:

• HLTF is frequently silenced by hypermethylation in colorectal cancer

• HLTF deficiency accelerates tumorigenesis in mouse models

• HLTF promotes error-free DNA repair, consistent with tumor suppressor function

Oncogenic properties:

• HLTF is upregulated in hepatocellular carcinoma and promotes tumor progression

• HLTF-deleted cancer cells show increased sensitivity to certain DNA damaging agents

• HLTF can activate specific signaling pathways like ERK/MAPK in HCC

Reconciliation approaches:

• Tissue-specific effects: HLTF may have different roles depending on tissue context

• Stage-specific functions: HLTF may be important for initial genome stability but problematic in established cancers

• Pathway dependencies: HLTF's function may depend on the status of other DNA repair pathways

• Consider both tumor cells and the tumor microenvironment, as HLTF status in both can affect cancer progression

What are the advantages and limitations of different HLTF knockdown/knockout approaches?

Researchers have multiple options for modulating HLTF expression:

How can researchers effectively study HLTF in primary human cells and tissues?

Working with HLTF in primary samples presents unique challenges:

Primary cell isolation and culture:

• HLTF is expressed in primary human macrophages and can be studied following monocyte differentiation

• Expression should be verified as it may vary with differentiation stage (e.g., detectable at day 4 or 7 post-differentiation)

• Primary lymphocytes and dendritic cells may also be relevant for studying HLTF's immune functions

Tissue sample analysis:

• Immunohistochemistry for HLTF requires careful optimization and validation

• FFPE samples can be used with appropriate antigen retrieval methods

• Spatial transcriptomics provides valuable information on positional gene patterns within intact tissue samples

Multi-omics approaches:

• Combining RNAseq with species-specific mapping and spatial transcriptomics provides comprehensive insights

• Proteomics approaches (e.g., 2D DIGE, MALDI-TOF/TOF mass spectrometry) complement transcriptomic data

• Integrated analysis yields holistic understanding of HLTF's role in complex biological processes

What are the most appropriate cellular assays for measuring HLTF-dependent DNA repair capacity?

To assess HLTF's impact on DNA repair mechanisms:

Replication stress tolerance:

• Colony formation assays following treatment with replication stressors (HU, MMS, MMC)

• HLTF-deficient cells show reduced DSB formation and increased survival upon replication stress

• HLTF loss confers mild resistance to alkylating agents like MNNG, but not to the level of MMR-deficient cells

DNA damage signaling:

• Western blot analysis of ATR/ATM activation markers following high-dose HU treatment (3mM)

• HLTF-KO cells show reduced DNA damage signaling compared to wild-type cells

• Analysis of γH2AX foci formation provides cellular resolution of DNA damage response

Pathway-specific assays:

• PRIMPOL-dependent mechanisms can be assessed in HLTF-KO cells using PRIMPOL knockdown/knockout

• REV1-dependent translesion synthesis in HLTF-HIRAN mutants requires specific TLS polymerase inhibition/depletion

• Combined genetic approaches (double knockouts, domain-specific mutations) help dissect pathway dependencies