Fig 7.

Protein-primed DNA synthesis activity of HP at low Mn²⁺ concentrations. Purified HP was washed twice before priming in TNK buffer with individual protease inhibitors and 10 mM β-ME to remove any EDTA present in the FLAG lysis buffer and then primed in the presence of [α-³²P]TTP and TMnNK buffer with the indicated concentrations of Mn²⁺. (A) Primed HP was visualized by SDS-PAGE and subsequent autoradiography. Lanes 5 to 8 represent a longer exposure of lanes 1 to 4. (B) HP priming products were mock treated (even-numbered lanes) or treated with Tdp2 (odd-numbered lanes, except for lane 1). The Tdp2-released nucleotides/DNA were resolved by urea-PAGE and visualized by autoradiography. The 5′-labeled 10-nt marker (Invitrogen) and DNA oligonucleotides (dTG, dTGA, and dTGAA) were loaded in lane 1, and their migration positions are indicated, as are the positions of TMP and TTP. (C) HP was incubated in a priming reaction mixture with the indicated concentrations of Mn²⁺ and/or Mg²⁺. Primed HP was visualized by SDS-PAGE and subsequent autoradiography.