Figure 2

Repair of linearized palsmids and dependence on TLK1, Rad9, and Ku70. A) Western blots of extracts depleted of TLK1, Rad9, and Ku70. B) Immunodepletion of TLK1 and add-back. The effect of immunodepleting the endogenous TLK1 in labeling of the ends and ligation/supercoiling is shown. In the middle lanes, recombinant TLK1B was added back to the immunodepleted extract in an amount comparable to the endogenous TLK1 (see panel A). Note that this gel was exposed for longer than that in Fig. 1C to reveal the repaired forms more clearly. Quantitation of the autorad in pixels is shown below each lane. C) Plasmid repair dependence on Rad9. Where indicated, the extract was immunodepleted of Rad9. In the left panel, we monitored repair of plasmid linearized with EcoRI alone (cohesive ends repair). In the middle panel, we monitored labeling and religation/supercoiling of plasmid cut with EcoRI/EcoRV, and its dependence on Rad9. In the right panel, Rad9 was added back. The position of linear and two supercoiled forms of the plasmids are indicated. D) Plasmid repair dependence on Ku70 and DNA-PK. The plasmid was in this case pre-labeled with Kleonw polymerase and [α-³²P]dATP. Where indicated, the extract was immunodepleted of Ku (left panel), or the reaction was carried out in presence of 1 μM wortmainnin (right panel). Where indicated, we added TLK1B.