Recombinant Human ATAD5 Protein: Properties & Research Uses

Recombinant Human ATAD5 Protein is a eukaryotic AAA+ ATPase critical for maintaining genome integrity by mediating PCNA unloading from DNA after replication and DNA damage repair.

Gene Name

ATAD5 (also known as ELG1 in yeast)

Protein Family

AAA+ ATPase, RFC-like clamp loader family

Primary Function

PCNA unloading from chromatin following DNA replication and repair

Biological Role

Maintains genome stability; regulates DNA damage tolerance and replication fork integrity

Expression System

Recombinant protein produced in a heterologous expression system

Research Applications

Biochemical assays, protein–protein interaction studies, DNA repair pathway research

Product SKU

rp367155

Biological Background and Function of ATAD5

ATAD5 (ATPase Family AAA Domain-Containing Protein 5) is a conserved eukaryotic protein that belongs to the AAA+ ATPase superfamily and shares structural homology with replication factor C (RFC) subunits. Its best-characterized function is the active unloading of proliferating cell nuclear antigen (PCNA) from chromatin upon completion of Okazaki fragment synthesis during DNA replication, as well as following DNA damage repair events.

PCNA is a homotrimeric sliding clamp that serves as a processivity factor for DNA polymerases and a platform for numerous DNA metabolism proteins. Precise regulation of PCNA loading and unloading is essential for genome integrity. ATAD5 forms an RFC-like complex (RLC-ATAD5) with RFC2–5 subunits and utilizes ATP hydrolysis to catalyze the opening and removal of the PCNA ring from double-stranded DNA.

Loss-of-function studies have demonstrated that depletion of ATAD5 leads to accumulation of chromatin-bound PCNA, increased genomic instability, replication stress, and hypersensitivity to DNA-damaging agents. ATAD5 mutations have been associated with elevated cancer susceptibility in human genetic studies, underscoring its importance as a tumor suppressor-like genome caretaker.

Biochemical Properties of Recombinant Human ATAD5 Protein

Recombinant Human ATAD5 Protein is produced using recombinant DNA technology in a defined heterologous expression system to ensure consistent batch-to-batch quality suitable for demanding biochemical applications. The protein retains the AAA+ ATPase domain architecture characteristic of the native human protein.

Key biochemical features include: ATP-binding and hydrolysis activity mediated by the conserved Walker A and Walker B motifs within the AAA domain; the capacity to interact with RFC2, RFC3, RFC4, and RFC5 to reconstitute the RLC-ATAD5 clamp-unloader complex in vitro; and direct binding affinity for homotrimeric PCNA. These properties make the recombinant protein suitable for in vitro ATPase activity assays, PCNA-binding and unloading assays, and co-immunoprecipitation or pull-down studies aimed at mapping protein–protein interactions within the DNA replication and repair machinery.

Researchers should store the protein according to the datasheet instructions, typically at –80 °C in a suitable buffer with a carrier protein such as BSA to prevent freeze–thaw degradation. Repeated freeze–thaw cycles should be avoided to maintain activity.

Research Applications

Recombinant Human ATAD5 Protein (SKU: rp367155) is a versatile reagent for multiple research contexts in DNA biology, cancer biology, and cell biology:

• In vitro PCNA Unloading Assays: The protein can be used to reconstitute the PCNA unloading reaction on model DNA substrates, enabling mechanistic dissection of clamp removal kinetics and ATP dependence.
• Protein–Protein Interaction Studies: Pull-down assays, surface plasmon resonance (SPR), and co-immunoprecipitation experiments can map ATAD5 interactions with RFC subunits, PCNA, and other replication/repair factors.
• ATPase Activity Measurement: Colorimetric or fluorometric phosphate release assays can characterize the intrinsic and DNA-stimulated ATPase activity of ATAD5.
• Structural Studies: Recombinant ATAD5 can serve as an antigen for antibody generation or as a component for cryo-EM and crystallographic studies of the RLC-ATAD5 complex.
• Drug Discovery Screening: As a genome-stability target implicated in cancer, ATAD5 is relevant to screening small-molecule inhibitors or activators of its ATPase or PCNA-interaction surfaces.

Handling, Storage, and Quality Considerations

To obtain optimal performance from Recombinant Human ATAD5 Protein, researchers should adhere to the following best-practice guidelines:

Storage: Upon receipt, centrifuge the vial briefly and store at –80 °C. Aliquot into single-use volumes before freezing to minimize freeze–thaw cycles, which can reduce ATPase activity and promote aggregation.

Buffer Compatibility: Ensure downstream assay buffers are compatible with the supplied formulation buffer. Dialysis or buffer exchange may be necessary if incompatible components are present.

Quality Control: Each lot of recombinant protein should be assessed for purity (typically by SDS-PAGE), identity (western blot with an anti-ATAD5 antibody), and functional activity (ATPase assay or PCNA-binding assay) before use in critical experiments.

Handling Precautions: Work on ice when manipulating the protein. Avoid vortexing; instead, mix by gentle pipetting. Use low-binding microtubes to minimize surface adsorption losses, particularly at low protein concentrations.

Expiry and Stability: Refer to the product datasheet for specific stability data. In the absence of empirical data, assume a working life of 6–12 months at –80 °C when stored correctly.

Frequently asked questions

What is the primary function of ATAD5 in human cells?

ATAD5 primarily functions as a PCNA unloader: it forms an RFC-like complex that uses ATP hydrolysis to remove the PCNA sliding clamp from chromatin after Okazaki fragment synthesis and DNA repair. This activity is essential for preventing aberrant PCNA accumulation, which would otherwise compromise genome stability.

What research applications is Recombinant Human ATAD5 Protein suitable for?

Recombinant Human ATAD5 Protein (rp367155) is suitable for in vitro PCNA unloading assays, ATPase activity measurements, protein–protein interaction studies (pull-down, co-IP, SPR), structural biology applications including cryo-EM, antibody production, and small-molecule drug discovery screening targeting genome-stability pathways.

How should Recombinant Human ATAD5 Protein be stored to maintain activity?

The protein should be stored at –80 °C immediately upon receipt. It is strongly recommended to aliquot the protein into single-use volumes prior to freezing to avoid repeated freeze–thaw cycles, which can reduce enzymatic activity and cause aggregation. Working on ice during experiments is advised.

Is ATAD5 relevant to cancer biology?

Yes. Human genetic studies have linked ATAD5 mutations and reduced expression to increased cancer susceptibility, positioning ATAD5 as a genome caretaker with tumor-suppressive properties. Its role in preventing replication stress and maintaining genome integrity makes it a relevant target in cancer research and potentially in therapeutic development.

What is the difference between ATAD5 and RFC1 in the context of PCNA regulation?

RFC1 is the large subunit of the canonical replication factor C complex (RFC1–5) responsible for loading PCNA onto DNA during replication. ATAD5, by contrast, replaces RFC1 in an alternative RFC-like complex (RLC-ATAD5) that performs the opposite function: unloading PCNA from DNA after replication or repair is complete. Both complexes share the RFC2–5 small subunits.

References

1. Kubota T, Nishimura K, Kanemaki MT, Donaldson AD. The Elg1 replication factor C-like complex functions in PCNA unloading during DNA replication. Mol Cell. 2013;50(2):273-280. doi:10.1016/j.molcel.2013.02.012
2. Lee KY, Fu H, Aladjem MI, Myung K. ATAD5 regulates the lifespan of DNA replication factories by modulating PCNA level on the chromatin. J Cell Biol. 2013;200(1):31-44. doi:10.1083/jcb.201206084
3. Shiomi Y, Nishitani H. Control of genome integrity by RFC complexes; conductors of PCNA loading onto and unloading from chromatin during DNA replication. Genes (Basel). 2017;8(1):52. doi:10.3390/genes8010052
4. Boerkoel CF, Takashima H, John J, et al. Mutant chromatin remodeling protein SMARCAL1 causes Schimke immuno-osseous dysplasia. Nat Genet. 2002;30(2):215-220.

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