Recombinant Human Treslin (TICRR), partial

What is Treslin/TICRR and what is its primary function in cells?

Treslin/TICRR (TopBP1-interacting replication stimulating protein/TopBP1-interacting checkpoint and replication regulator) is a protein required for the initiation of DNA replication in eukaryotic cells. It functions as part of a multiprotein complex that helps convert licensed replication origins into active replication forks. Mechanistically, Treslin/TICRR coordinates the recruitment of essential replication factors to origins of replication.

The protein's primary role is mediating the loading of Cdc45 onto chromatin, which is a critical step for the activation of the CMG (Cdc45-MCM-GINS) helicase complex. This activation allows for the unwinding of DNA and subsequent DNA synthesis. Experimental evidence shows that immunodepletion of Treslin-MTBP from Xenopus egg extracts severely impairs DNA replication, demonstrating its essential role in this process . Additionally, studies have confirmed that Treslin collaborates with TopBP1 in triggering DNA replication initiation .

The recruitment of Treslin to chromatin begins before S phase and continues during S phase progression, indicating its dynamic role throughout the replication process. This recruitment pattern suggests that Treslin/TICRR not only helps initiate replication but may also play a role in regulating the timing of origin firing across S phase .

What is the domain architecture of Treslin/TICRR?

Treslin/TICRR exhibits a complex domain architecture with both conserved and higher eukaryote-specific regions. The essential core of the protein consists of three key domains: the M domain, the STD (Sld3-like domain), and the TDIN domain, which are flanked by higher eukaryote-specific terminal domains .

The M domain is responsible for binding to MTBP (Mdm2 Binding Protein), which is the metazoan homolog of yeast Sld7. This interaction is crucial for Treslin's function in replication initiation. The STD domain shows sequence-based evidence for homology with yeast Sld3 and is essential for replication, as demonstrated through RNAi-replacement experiments in human cells. Although the precise molecular function of the STD domain remains incompletely characterized, its homology with Sld3 suggests it might support origin firing through interaction with Cdc45 .

The TDIN domain is a conserved region containing two CDK phosphorylation sites that mediate binding to TopBP1. Specifically, phosphorylation of S1001 within this domain creates a binding site for the BRCT domains of TopBP1, forming a critical interaction required for replication initiation . Mutations of these phosphorylation sites significantly reduce the interaction with TopBP1 and result in deficient DNA replication .

RNAi-replacement experiments have confirmed that all three of these domains—M, STD, and TDIN—perform essential functions during origin firing in human cells. Deletion of the STD domain results in only 30-50% replication efficiency compared to wild-type Treslin, highlighting its importance .

How does Treslin/TICRR interact with MTBP and what is the stoichiometry of this complex?

Treslin/TICRR forms a complex with MTBP that is critical for DNA replication initiation. Biochemical studies using Xenopus egg extracts have revealed that MTBP and Treslin form an elongated tetramer containing two molecules of each protein, resulting in a 2:2 stoichiometry .

Immunoprecipitation experiments with MTBP antibodies co-precipitated Treslin, and total protein staining of the immunoprecipitates showed major bands at the expected positions of MTBP and Treslin. Mass spectrometry confirmed that MTBP and Treslin were the major protein components of the immunoprecipitate .

The interaction between Treslin and MTBP is mediated through Treslin's M domain, which is essential for MTBP binding. Functional studies have shown that MTBP alone is not sufficient to rescue replication in MTBP-depleted extracts, indicating that the Treslin-MTBP complex, rather than MTBP alone, is required for DNA replication . This suggests that the two proteins function as an obligate complex during replication initiation.

What methods can be used to purify recombinant Treslin-MTBP complex for functional studies?

Purification of the Treslin-MTBP complex for functional studies requires a multi-step approach that preserves the integrity of the complex while achieving sufficient purity and yield. Based on successful purification strategies from Xenopus egg extracts, the following methodological approach is recommended:

First, start with clarified supernatant of whole egg extract or an appropriate expression system for recombinant proteins. Precipitate proteins using polyethylene glycol (PEG), which serves as an initial concentration step. In the documented purification from Xenopus egg extracts, this step maintained the Treslin-MTBP complex while beginning to remove contaminant proteins .

Next, apply sequential chromatography steps, beginning with cation exchange chromatography. This technique separates proteins based on their positive charge interactions with the negatively charged resin. Follow this with anion exchange chromatography, which separates proteins based on their negative charge interactions with the positively charged resin. This combination of ion exchange steps has proven effective in enriching the Treslin-MTBP complex .

For final concentration and buffer exchange, ammonium sulfate precipitation can be employed, though it's important to note that residual ammonium sulfate can inhibit DNA replication in functional assays . Therefore, thorough dialysis or buffer exchange is essential before using the purified complex in functional experiments.

When executed properly, this purification strategy can achieve a greater than 600-fold enrichment of both Treslin and MTBP while maintaining their complex integrity. Mass spectrometry analysis of the purified material should confirm that Treslin and MTBP are the major protein components .

For functional verification of the purified complex, rescue experiments in immunodepleted extracts provide a reliable assay. In Xenopus egg extract studies, the 600-fold enriched Treslin-MTBP complex rescued replication to 60% compared to positive controls, confirming the functionality of the purified complex .

How can researchers experimentally distinguish between DDK and CDK regulation of Treslin/TICRR?

Distinguishing between DDK (Dbf4-dependent kinase) and CDK (Cyclin-dependent kinase) regulation of Treslin/TICRR requires careful experimental design using selective inhibitors, phosphorylation site mutants, and biochemical assays. Here are methodological approaches to differentiate their effects:

One effective approach is using selective kinase inhibitors. For DDK inhibition, researchers can employ compounds such as PHA-767491, TAK-931 (simurosertib), or XL413. Each has varying specificity and potency; TAK-931 has been characterized as a more specific but marginally less potent inhibitor of DDK-mediated chromatin-associated Mcm4 phosphorylation compared to PHA-767491 . For CDK inhibition, p27KIP1 or specific CDK inhibitors can be used. By applying these inhibitors individually or in combination, researchers can parse out the distinct contributions of each kinase to Treslin regulation.

The mobility shift assay is particularly informative for distinguishing phosphorylation by different kinases. Treslin exhibits characteristic migration patterns on SDS-PAGE depending on its phosphorylation state. Inhibition of either DDK or CDK activity alone causes Treslin to migrate faster than in control conditions, but to different extents. When both DDK and CDK are inhibited simultaneously, Treslin migrates even faster than with each inhibition alone, suggesting independent contributions from both kinases to Treslin phosphorylation .

Phosphorylation site-specific antibodies or mass spectrometry can be employed to identify which sites are phosphorylated under different conditions. For CDK regulation, S1001 is a well-characterized site whose phosphorylation mediates interaction with TopBP1 . Researchers can create phosphomimetic or non-phosphorylatable mutants of specific sites to determine their contribution to Treslin function.

Protein-protein interaction assays using co-immunoprecipitation or pulldown experiments help determine how each kinase affects Treslin's interactions with partners such as TopBP1 and chromatin. Experiments have shown that DDK inhibition by PHA-767491 or TAK-931 reduces the interaction of Treslin with TopBP1, even in the presence of optimal CDK activity . This effect can be partially reversed by adding the PP1 inhibitor Tautomycetin, suggesting that PP1 phosphatase antagonizes DDK-mediated phosphorylation events .

What experimental approaches can be used to study the chromatin association dynamics of Treslin/TICRR during cell cycle progression?

Studying the chromatin association dynamics of Treslin/TICRR during cell cycle progression requires specialized techniques that can detect protein-chromatin interactions temporally. Here are methodological approaches for such studies:

Chromatin isolation and immunoblotting provide a straightforward approach to examine Treslin's association with chromatin at different cell cycle stages. Cell fractionation protocols that separate soluble proteins from chromatin-bound proteins, followed by immunoblotting for Treslin, can reveal temporal patterns of association. In Xenopus egg extracts, this technique has shown that Treslin-MTBP is recruited onto chromatin before S phase starts, and recruitment continues during S phase progression .

Cell synchronization methods allow researchers to study chromatin association at defined cell cycle stages. This can be achieved using chemical synchronization (thymidine block, nocodazole, etc.) or physical methods (mitotic shake-off). After synchronization, cells can be released and sampled at various time points to track Treslin's chromatin association through G1, S, G2, and M phases.

Immunofluorescence microscopy provides spatial information about Treslin localization. By co-staining for Treslin alongside markers of replication (EdU, PCNA) and using cell cycle markers, researchers can visualize the association of Treslin with chromatin in individual cells across the cell cycle.

For more detailed mechanistic studies, chromatin immunoprecipitation (ChIP) can be employed to identify specific genomic regions where Treslin binds. This can be coupled with sequencing (ChIP-seq) to obtain genome-wide binding profiles at different cell cycle stages.

To investigate the requirements for Treslin's chromatin association, researchers can manipulate specific factors and observe the effects on Treslin binding. For instance, studies in Xenopus egg extracts have shown that Treslin-MTBP chromatin association is strongly dependent on origin licensing (MCM2-7 loading), with geminin treatment (which inhibits licensing) reducing Treslin-MTBP chromatin binding to approximately 20% of control levels . Similarly, DDK activity increases and strengthens Treslin-MTBP interaction with licensed chromatin, while CDK activity reduces this interaction .

Functional assays can determine whether the Treslin bound to chromatin under different conditions is biochemically active. For example, researchers isolated chromatin from extracts treated with geminin and tested its ability to replicate in MTBP-depleted extract. The chromatin replicated to less than 20% of control levels, suggesting that origin licensing is required for Treslin-MTBP to be efficiently recruited to chromatin in a functional manner .

How do CDK and DDK activities coordinate to regulate Treslin/TICRR function during replication initiation?

CDK (Cyclin-dependent kinase) and DDK (Dbf4-dependent kinase) activities coordinate in a sophisticated manner to regulate Treslin/TICRR function during replication initiation. This dual kinase regulation ensures proper timing and efficiency of DNA replication initiation.

CDK activity primarily regulates Treslin's interaction with TopBP1, which is crucial for pre-initiation complex formation. CDK-dependent phosphorylation of the well-conserved S1001 residue in Treslin's TDIN domain mediates its interaction with the phospho-binding BRCT domains of TopBP1 . Mutation of this residue significantly reduces the interaction of Treslin with TopBP1 and results in deficient DNA replication. Cells expressing a phosphomimetic mutant of Treslin display faster replication kinetics and a shorter S phase, further confirming the importance of this CDK-mediated phosphorylation .

DDK activity plays multiple roles in regulating Treslin function. First, DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin . Second, DDK activity is required for the CDK-dependent interaction between Treslin-MTBP and TopBP1, representing a critical regulatory step in pre-initiation complex formation. Inhibition of DDK activity using inhibitors like PHA-767491 or TAK-931 restricts the interaction between Treslin-MTBP and TopBP1, even in the presence of optimal CDK activity to support complex formation .

The interplay between these kinases creates a regulatory circuit. While both licensing and DDK activity are required to increase and strengthen the interaction of Treslin-MTBP with chromatin, CDK activity reduces this interaction . This suggests that as CDK activity rises during S phase, Treslin-MTBP is likely recruited to replication origins as part of a complex with TopBP1, rather than binding directly to chromatin . This mechanism may help ensure that replication initiates only at licensed origins and only when both CDK and DDK activities have reached appropriate levels.

Additionally, this dual regulation is subject to reversal by protein phosphatase 1 (PP1). The inhibition of Treslin-TopBP1 interaction by DDK inhibitors can be partially reversed by the addition of the PP1 inhibitor Tautomycetin, consistent with the role of PP1 in reversing DDK-mediated phosphorylation .

What are the consequences of Treslin/TICRR depletion or mutation on DNA replication and cell cycle progression?

The consequences of Treslin/TICRR depletion or mutation on DNA replication and cell cycle progression are profound, reflecting its essential role in replication initiation. Multiple experimental approaches have revealed specific molecular and cellular outcomes of Treslin dysfunction.

Immunodepletion of Treslin-MTBP from Xenopus egg extracts results in severe inhibition of DNA replication. Add-back experiments demonstrated that Treslin-MTBP is rate-limiting for replication initiation, as addition of just 5% by volume of undepleted extract (corresponding to approximately 0.05 nM Treslin-MTBP tetramer) could rescue replication to about 50% efficiency in depleted extracts . This rate-limiting property suggests that Treslin levels may be a critical determinant of replication timing and efficiency.

At the molecular level, Treslin-MTBP depletion blocks the assembly of the pre-initiation complex (pre-IC) required for CMG helicase formation. In both MTBP-depleted and Treslin-depleted extracts, TopBP1 is recruited to chromatin, but the recruitment of downstream factors including RecQ4, Cdc45, and PCNA is strongly reduced . This indicates that Treslin-MTBP functions at a specific step in the replication initiation pathway, after TopBP1 recruitment but before Cdc45 loading and CMG helicase activation.

The interaction between Treslin and TopBP1 is particularly critical for replication. Mutation of the CDK phosphorylation site S1001 to a non-phosphorylatable form significantly reduces the interaction with TopBP1 and results in deficient DNA replication . Conversely, cells expressing a phosphomimetic Treslin mutant display faster replication kinetics and a shorter S phase, demonstrating that modulation of Treslin function can directly impact S phase progression .

Domain-specific mutations have revealed that multiple regions of Treslin are essential for its function. Deletion of the STD domain, which shows homology to yeast Sld3, reduces replication efficiency to only 30-50% compared to wild-type Treslin . Similarly, mutations in the M domain (MTBP-binding) and TDIN domain (TopBP1-binding) also impair replication, confirming that all three domains are required for full Treslin function .

How does the Treslin-MTBP-TopBP1 interaction network function in replication origin selection and activation?

The Treslin-MTBP-TopBP1 interaction network plays a central role in replication origin selection and activation through a series of regulated protein-protein interactions that couple origin licensing to initiation. This network functions as a molecular switch that helps determine which licensed origins will fire and when they will do so during S phase.

The process begins with the loading of MCM2-7 helicase complexes onto origin DNA during G1 phase, which licenses these sites for potential replication. Treslin-MTBP associates with these licensed origins in a manner that depends on both the presence of MCM2-7 and DDK activity . This association is strengthened by DDK-mediated phosphorylation of MCM proteins, particularly Mcm4, creating a preferential binding platform for Treslin-MTBP at licensed origins .

As cells enter S phase and CDK activity increases, Treslin undergoes CDK-dependent phosphorylation at S1001, which creates a binding site for the BRCT domains of TopBP1 . This phosphorylation actually reduces Treslin's affinity for MCM2-7 while increasing its affinity for TopBP1, suggesting a handoff mechanism . Importantly, this CDK-dependent interaction between Treslin and TopBP1 also requires DDK activity, creating a dual-kinase regulatory checkpoint for origin activation .

The formation of the tripartite Treslin-MTBP-TopBP1 complex is a crucial step for the subsequent recruitment of Cdc45 and the GINS complex, leading to the formation of the active CMG (Cdc45-MCM-GINS) helicase . Experimental evidence supports this model, as depletion of Treslin or MTBP prevents the recruitment of Cdc45 to chromatin, while TopBP1 recruitment is maintained albeit at reduced levels .

The temporal regulation of this network contributes to the ordered firing of origins throughout S phase. TopBP1 chromatin association follows that of Treslin-MTBP, with a peak of binding during S phase that declines as S phase proceeds . In extracts depleted of Treslin-MTBP, the association of TopBP1 with chromatin is reduced but does not decline at later times, suggesting that Treslin-MTBP influences not only the initial recruitment of TopBP1 but also its dynamics during S phase progression .

This interaction network provides multiple regulatory points where cellular signaling can influence origin selection and activation. For example, the PP1 phosphatase can reverse DDK-mediated phosphorylation events, potentially allowing for dynamic regulation of origin firing in response to replication stress or other cellular conditions .

What are the most effective systems for studying Treslin/TICRR function in vitro and in vivo?

Research on Treslin/TICRR employs several complementary experimental systems, each with distinct advantages for investigating different aspects of Treslin function. Understanding the strengths and limitations of these systems is crucial for designing effective experiments.

Xenopus egg extracts represent a powerful in vitro system for studying Treslin function in DNA replication. This cell-free system recapitulates the entire process of DNA replication and allows for straightforward manipulation through addition or depletion of proteins. The system has been instrumental in establishing the role of Treslin-MTBP as a rate-limiting factor for replication initiation and in characterizing its interactions with chromatin and other replication factors . A key advantage is the ability to control cell cycle stage by manipulating cyclin levels, allowing researchers to study Treslin function at precise points in the cell cycle. Additionally, the high concentration of Treslin in egg extracts facilitates biochemical purification and characterization of the native protein complex .

Human cell culture systems provide an important in vivo context for Treslin studies. RNAi-replacement approaches, where endogenous Treslin is depleted by siRNA and replaced with mutant versions, have been particularly valuable for structure-function analyses . This approach has revealed the importance of specific domains like the STD domain for Treslin function in human cells . Human cells also allow for studies of Treslin regulation throughout the normal cell cycle and in response to replication stress, providing physiological relevance that complement in vitro findings.

Recombinant protein expression systems enable the production and purification of Treslin fragments or full-length protein for biochemical and structural studies. While expressing full-length Treslin has proven challenging due to its large size and complex structure, expression of specific domains has been more successful and has contributed to our understanding of Treslin's domain architecture and function . These systems also facilitate the creation of site-specific mutations to test the importance of particular residues or motifs.

For investigating evolutionary aspects of Treslin function, comparative studies across species can be highly informative. The identification of Treslin as the metazoan homolog of yeast Sld3, despite limited sequence conservation, has provided important insights into the evolution of replication initiation mechanisms . Such comparative approaches can help distinguish conserved core functions from species-specific adaptations.

What strategies can be used to create and validate phospho-specific mutants of Treslin/TICRR?

Creating and validating phospho-specific mutants of Treslin/TICRR requires careful experimental design to ensure both molecular and functional characterization. The following methodological approach outlines strategies for generating and validating such mutants:

Site selection should be guided by sequence analysis and existing knowledge. For Treslin, S1001 has been identified as a critical CDK phosphorylation site mediating TopBP1 interaction . Researchers should identify consensus phosphorylation motifs (S/T-P for CDKs) and consider evolutionary conservation across species to prioritize sites. Mass spectrometry analysis of purified Treslin can also identify phosphorylated residues in vivo.

For mutagenesis, two common approaches are used: non-phosphorylatable mutations (typically S/T to A) and phosphomimetic mutations (S/T to D/E). For example, converting S1001 to alanine prevents phosphorylation, while conversion to aspartic or glutamic acid mimics constitutive phosphorylation. These complementary approaches help distinguish between the importance of the residue itself versus its phosphorylation status.

Expression systems must be carefully chosen. For cellular studies, researchers have successfully used RNAi-replacement systems where endogenous Treslin is depleted by siRNA and replaced with siRNA-resistant mutant versions . For biochemical studies, recombinant expression of Treslin fragments containing the relevant phosphorylation sites may be more manageable than full-length protein.

Molecular validation should confirm that the mutations affect phosphorylation status as expected. This can be achieved through phospho-specific antibodies (if available), Phos-tag gels that enhance mobility shifts of phosphorylated proteins, or mass spectrometry. For Treslin, mobility shift assays on standard SDS-PAGE have successfully detected differences in phosphorylation status, with hypophosphorylated forms migrating faster than phosphorylated forms .

Interaction assays are essential to validate functional consequences of phosphorylation. Co-immunoprecipitation or pulldown assays can determine whether phospho-mutants affect interactions with known partners like TopBP1. For instance, S1001A mutation in Treslin significantly reduces its interaction with TopBP1 .

Functional validation through replication assays is crucial to determine the biological significance of the phosphorylation sites. BrdU incorporation assays can measure replication efficiency in cells expressing phospho-mutants compared to wild-type Treslin . In Xenopus egg extracts, measuring DNA replication by incorporation of radiolabeled nucleotides provides quantitative data on how phospho-mutants affect replication .

Timing and cell cycle studies can reveal whether phosphorylation affects the temporal regulation of Treslin function. Cells expressing phosphomimetic Treslin mutants have been shown to display faster replication kinetics and a shorter S phase, indicating that phosphorylation can influence the timing of replication .

How can researchers quantitatively assess the impact of Treslin/TICRR mutations on protein-protein interactions and DNA replication?

Quantitative assessment of Treslin/TICRR mutations requires rigorous experimental approaches that can measure both biochemical interactions and functional outcomes with precision. The following methodological strategies provide a comprehensive framework for such analyses:

For protein-protein interaction quantification, co-immunoprecipitation (co-IP) followed by immunoblotting provides a semi-quantitative measure of interactions between Treslin mutants and binding partners like TopBP1 or MTBP. For more precise quantification, researchers can use densitometry to analyze band intensities, comparing the ratio of co-precipitated protein to immunoprecipitated bait across different samples . To control for expression level differences, input samples should be analyzed alongside immunoprecipitates, and results can be expressed as a percentage of the wild-type interaction.

Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) offers more precise biophysical measurements of binding affinities between purified proteins. These techniques can determine binding constants (Kd values) for wild-type and mutant Treslin interactions with partners, providing quantitative measures of how mutations affect binding energetics.

For chromatin association quantification, chromatin fractionation followed by immunoblotting can measure the amount of Treslin mutants bound to chromatin. Comparing these values to wild-type Treslin under identical conditions provides a relative measure of chromatin binding efficiency. In Xenopus egg extracts, researchers have used this approach to show that DDK activity increases and strengthens Treslin-MTBP interaction with chromatin .

DNA replication assays provide functional readouts of Treslin mutant activity. In cell-based systems, BrdU incorporation measured by flow cytometry or microscopy can quantify replication efficiency. The results can be expressed as percentage of replicating cells or as intensity of BrdU signal relative to wild-type controls . RNAi-replacement systems where endogenous Treslin is depleted and replaced with mutant versions are particularly valuable for these analyses.

In Xenopus egg extracts, replication can be quantified by measuring incorporation of radiolabeled nucleotides into newly synthesized DNA. This approach has revealed that the Treslin-MTBP complex is rate-limiting for replication, with a specific relationship between Treslin-MTBP concentration and replication efficiency . Similar dose-response experiments with mutant proteins can determine how mutations affect this relationship.

Rescue experiments provide a powerful approach to quantify the functional impact of mutations. By adding back wild-type or mutant Treslin to depleted extracts or cells, researchers can measure the ability of each protein to restore replication. For example, studies have shown that while Treslin-MTBP complex can rescue replication in MTBP-depleted extract to approximately 30% of the positive control, MTBP alone cannot rescue replication at all .

To relate biochemical interactions to functional outcomes, researchers should perform correlation analyses between interaction strength (measured by co-IP or biophysical methods) and replication efficiency (measured by BrdU incorporation or similar assays). This can reveal whether the functional defects observed with certain mutations are directly proportional to their effects on specific protein interactions.

What are the most promising approaches for determining the high-resolution structure of Treslin/TICRR?

Domain-by-domain structural analysis represents a practical approach for Treslin, given its modular architecture with distinct functional domains. The M domain (MTBP-binding), STD domain (Sld3-like), and TDIN domain (TopBP1-binding) could be individually expressed, purified, and subjected to structural studies . This approach has proven successful for many large, multi-domain proteins where full-length structures are challenging to obtain.

For X-ray crystallography, researchers should focus on well-folded, soluble domains of Treslin in complex with their binding partners. For example, co-crystallization of the TDIN domain with the relevant BRCT domains of TopBP1 could reveal the structural basis of their phosphorylation-dependent interaction. Crystallization of such complexes often benefits from limited proteolysis to remove flexible regions that might hinder crystal formation.

Cryo-electron microscopy (cryo-EM) offers particular promise for larger assemblies, such as the complete Treslin-MTBP tetramer or the tripartite Treslin-MTBP-TopBP1 complex. The recent advances in cryo-EM technology allowing near-atomic resolution of smaller complexes make this approach increasingly feasible. Biochemical evidence suggests that MTBP forms an elongated tetramer with Treslin containing two molecules of each protein , providing a suitable target for cryo-EM analysis.

Integrative structural biology combining multiple techniques could prove especially valuable for Treslin. This approach might include low-resolution shapes from small-angle X-ray scattering (SAXS) or negative-stain EM, distance constraints from crosslinking mass spectrometry, and high-resolution structures of individual domains from X-ray crystallography or NMR spectroscopy. Computational modeling can then integrate these diverse data to generate comprehensive structural models.

For protein production, the choice of expression system is critical. While E. coli expression might work for some isolated domains, eukaryotic expression systems such as insect cells or mammalian cells are likely necessary for larger fragments or full-length Treslin to ensure proper folding and post-translational modifications. The successful purification of Treslin-MTBP from Xenopus egg extracts suggests that native sources might also provide material for structural studies .

Protein engineering strategies may help overcome difficulties in expression and crystallization. These include the use of truncations guided by secondary structure predictions, surface entropy reduction (replacing flexible, charged surface residues with alanines), and fusion to crystallization chaperones like T4 lysozyme or BRIL.

What are the key unresolved questions about Treslin/TICRR regulation and function in replication?

Despite significant progress in understanding Treslin/TICRR, several key questions remain unresolved regarding its regulation and function in DNA replication initiation:

The complete phosphoregulation map of Treslin remains to be established. While S1001 has been identified as a critical CDK target for TopBP1 binding , Treslin likely contains numerous other phosphorylation sites targeted by various kinases. Evidence suggests that Treslin is a target of DDK activity , but the specific DDK phosphorylation sites and their functional significance remain incompletely characterized. A comprehensive phosphorylation site mapping and functional analysis would significantly advance our understanding of Treslin regulation.

The molecular mechanism by which Treslin-MTBP contributes to Cdc45 loading onto MCM2-7 requires further elucidation. Based on homology with yeast Sld3, Treslin's STD domain might mediate interaction with Cdc45 , but the structural and biochemical details of this interaction in metazoans remain to be determined. Understanding this process is crucial, as Cdc45 loading is a key step in CMG helicase activation.

The precise architecture of the pre-initiation complex (pre-IC) and how Treslin-MTBP orchestrates its assembly remains incompletely understood. While we know that Treslin-MTBP interacts with TopBP1 and associates with chromatin-bound MCM2-7, the spatiotemporal dynamics of these interactions and how they coordinate the recruitment of other factors like GINS, RecQ4, and DNA polymerases require further investigation.

The role of Treslin in replication origin selection and the timing of origin firing remains an open question. Given that Treslin-MTBP is rate-limiting for replication initiation , it may play a key role in determining which licensed origins fire and when they do so during S phase. The mechanisms controlling the distribution of Treslin-MTBP among potential origins and how this contributes to the replication timing program need further exploration.

The function of Treslin beyond canonical replication initiation deserves investigation. Given its interactions with multiple replication and checkpoint proteins, Treslin may play roles in replication stress responses, DNA damage signaling, or cell cycle checkpoints that are currently underappreciated. The observation that Treslin interacts with TopBP1, which has dual roles in replication and checkpoint signaling , suggests potential broader functions.

The regulation of Treslin-MTBP complex assembly and stability throughout the cell cycle is not fully understood. While we know that MTBP and Treslin form a 2:2 tetramer , the factors controlling complex formation, potential post-translational modifications affecting complex stability, and whether the complex composition changes under different cellular conditions remain to be determined.

How might understanding Treslin/TICRR function contribute to approaches for targeting DNA replication in cancer therapy?

Understanding Treslin/TICRR function opens new avenues for targeting DNA replication in cancer therapy, potentially leading to more selective and effective therapeutic strategies. The current knowledge about Treslin suggests several promising approaches:

Targeting the rate-limiting property of Treslin-MTBP could provide a selective advantage for cancer therapy. Since Treslin-MTBP is present at limiting levels for replication initiation , cancer cells with elevated replication rates may be particularly dependent on Treslin function. Small molecules that disrupt Treslin-MTBP complex formation or its interactions with other replication factors could selectively impact rapidly proliferating cancer cells while sparing normal cells with lower replication demands.

The phosphorylation-dependent interactions of Treslin offer targetable nodes for intervention. The CDK-dependent interaction between Treslin and TopBP1, mediated by phosphorylation of S1001 , represents a potential target for small molecule inhibitors. Similarly, the DDK-dependent strengthening of Treslin-MTBP association with chromatin could be disrupted by appropriate inhibitors. Compounds that mimic phosphopeptides or occupy phosphopeptide-binding pockets could be developed to interfere with these critical interactions.

Synthetic lethality approaches could exploit Treslin's functions in relation to other replication or repair pathways. Cancer cells with defects in specific DNA repair pathways or checkpoint mechanisms might be particularly vulnerable to partial inhibition of Treslin function. For example, cells with compromised homologous recombination (like BRCA-deficient cancers) might show enhanced sensitivity to agents that disrupt Treslin-dependent replication initiation.

Combination therapy strategies could target different aspects of the replication initiation pathway. Since DDK and CDK activities cooperate to drive the interaction of Treslin-MTBP with TopBP1 , combining inhibitors of both kinases at lower doses might achieve synergistic effects while reducing toxicity. Similarly, combining Treslin-targeting approaches with existing replication inhibitors (like gemcitabine or hydroxyurea) could enhance therapeutic efficacy.

Structure-based drug design approaches will benefit from advances in understanding Treslin's molecular architecture. As high-resolution structures of Treslin domains and their complexes become available, rational design of small molecules targeting specific protein-protein interfaces will become feasible. Of particular interest would be the interfaces between Treslin and MTBP, Treslin and TopBP1, or Treslin and MCM2-7.

Biomarker development based on Treslin activity or expression could help identify patients most likely to respond to replication-targeting therapies. Since Treslin-MTBP is rate-limiting for replication , its expression levels or activity state might predict sensitivity to certain therapeutic approaches. Developing assays to measure Treslin function in patient samples could aid in treatment selection and monitoring.