Figure 3. Efficient Resection of the Nascent Lagging Strand Is Impaired when CMG Unloading Is Blocked.

(A) pICL plasmid was replicated in NPE containing [α-³²P] dATP, and repair intermediates were separated on a native agarose gel and visualized by autoradiography. OC, open circular; SC, supercoiled.

(B) pICL was replicated in extract with [α-³²P]dATP. 15 min after initiation of replication, extract was supplemented with cytosine arabinoside triphosphate (araCTP) (2 mM) or aphidicolin (50 μM). Replication intermediates were separated on a native agarose gel after deproteinization and visualized by autoradiography. Red arrowhead, slow figure 8; Blue arrowhead, fast figure 8; green arrowhead, reversed forks. To determine the absolute fraction of reversed forks, the radioactivity adjacent to the green bar in lane 3 was divided by the total radioactivity in the lane. To determine the fraction of pre-incision intermediates comprising reversed forks, the radioactivity in the green bar was divided by the radioactivity adjacent to the pink bar. The gel is representative of three independent experiments.

(C) Model depicting the rightward stalled fork in the presence of LacR, together with the BsaI site and primer used to generate the sequencing ladder.

(D) Same as (C) but in the presence of p97i.

(E) pICL^LacO was replicated with [³²P-α]dATP in the presence of LacR or p97i, and nascent strand products were analyzed by denaturing PAGE after digestion with BsaI. Red arrow, stalled leading strand; orange line, lagging strands of the rightward fork. The sequencing ladder was generated with primer R (C) and (D). Similar results were obtained in a second, independent experiment.