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. 2012 May 14;40(15):7430–7441. doi: 10.1093/nar/gks416

Figure 2.

Figure 2.

Assay for site-specific UV cross-linking of DNA primers to the TER subunit in active Tetrahymena telomerase holoenzyme complexes. (A) Schematic drawing of the cross-linking assay (see the text for a detailed description). Only one of several telomerase accessory proteins is shown here for clarity. S denotes a 4-thio dT substitution. Small g denotes a radioactively labeled G residue added to the DNA primer by the cross-linked enzyme. The star indicates the active site. (B) Control assays performed with the primer S-5 6-mer shown in A and with an unsubstituted derivative (see ‘Materials and Methods’ section for primer nomenclature). Cross-linked complexes were prepared by the procedure illustrated in A. The complexes were analyzed by SDS–PAGE, followed by phosphorimaging. Lane 1: complete assay. Lane 2: same primer with no substitution. Lane 3: RNase A treatment before irradiation (0.02 mg/ml RNase A, 45 min incubation at 37°C). Lane 4: RNase A treatment after primer extension (0.02 mg/ml RNase A, 45 min incubation at 37°C). Lane 5: proteinase K treatment after primer extension (0.2 mg/ml proteinase K, 1.5 hr incubation at 37°C in the presence of 0.25% SDS). The arrow indicates the cross-linked TER–primer. (C) Control assays performed with the primer S-20 21-mer and with an unsubstituted derivative. Procedures and lane assignments are the same as in B.