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. Author manuscript; available in PMC: 2017 Oct 3.
Published in final edited form as: Nat Cell Biol. 2017 Apr 3;19(5):468–479. doi: 10.1038/ncb3500

Figure 6. CUL2LRR1 associates with the post-termination vertebrate replisome and is recruited to chromatin during DNA replication termination in Xenopus egg extracts.

Figure 6

(a) Experimental scheme for isolation of proteins associated with the CMG helicase after termination in the absence of replisome disassembly, in extracts of Xenopus laevis eggs. (b) Immunoblots of input and the indicated IP samples for the experiment in (a). (c) Replisome disassembly was inhibited with the p97 inhibitor NMS873, and LRR1 was then isolated from digested chromatin at the 70’ timepoint, in parallel with a control IP with IgG, before detection of the indicated proteins by immunoblotting. (d) Chromatin association of the indicated factors was monitored by immunoblotting, at the indicated timepoints after addition of sperm chromatin to egg extracts (except for the -DNA sample that lacked chromatin). Where indicated, replication initiation was blocked by addition of p27(KIP1) or Geminin. The neddylase inhibitor MLN4924 was added to all samples to block replisome disassembly. (e) Timecourse experiment comparing chromatin-bound factors in the absence or presence of the neddylation inhibitor MLN4924. (f) Replication kinetics were monitored for the experiment in (e), by incorporation of radiolabelled α-dATP into newly synthesised DNA (see also Supplementary Figure 5b; source data for repeats of this experiment are included in Supplementary Table 6). (g) Inhibition of DNA synthesis blocks association of CUL2LRR1 with chromatin. DNA synthesis was inhibited with the DNA polymerase inhibitor aphidicolin. Caffeine was added to inactivate the S-phase checkpoint, which otherwise would have reduced the level of CMG on chromatin +Aphidicolin. (h) Analogous experiment to that in (e), showing that CUL2-LRR1 accumulated on chromatin with CMG when replisome disassembly was blocked by the p97 inhibitor NMS873, but chromatin recruitment of CUL2-LRR1 was inhibited if DNA replication termination was delayed by addition of the TOPO2 inhibitor ICRF193. Unprocessed scans of key immunoblots are shown in Supplementary Figure 8.