TDP1

Product Description

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TopoGEN now provides purified Human Tyrosyl DNA Phosphodiesterase (Tdp1) purified to homogeneity to a single 68 kDa band on SDS-PAGE. Tdp1 is catalytically active and will remove a terminal 3’ phosphotyrosyl group from a specifically designed oligo target site (oligo-Tyr). Additionally, TopoGEN scientists have demonstrated that Tdp1 will release topo I covalently attached to genomic DNA isolated from cells treated with camptothecin using TopoGEN’s In Vivo Link Kit. Tdp1 is thought to be important in repair of topo/DNA adducts that form following topo-active drug therapy; therefore, there is considerable interest in identifying drugs that target Tdp1. Such drugs may work well in combination with topo targeting strategies.

Figure 1. SDS-PAGE of TopoGEN Purified TDP1.
The gel was intentionally overloaded with 20 ug of protein to reveal
minor breakdown products.

Unit Defintion

One unit of Tdp1 can release the terminal Tyrosine from 20 ng of substrate (oligo-Tyr) in 30 min at 37^oC.

Tdp1 Reaction.

Release of Topo I from the covalent complex is shown In Fig. 2. Figure 2. Activity of Tdp-1

Tdp1 Assays

Biochemical assays for Tdp1 can be carried out using an oligo nucleotide with a 3’ terminal tyrosine residue. Release of tyrosine from a 5’ 32P end labeled oligo is easily measured using a denaturing polyacrylamide gel or DNA sequencing gels. TopoGEN Provides a kit for Tdp1 assay that has key reagents and controls/protocols for setting up a complete assay for Tdp1; see Catalog # 1004.

Schematic of Tdp1 activity. Tdp1 releases topo I from DNA as shown in the outline above.