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. Author manuscript; available in PMC: 2015 Jan 26.
Published in final edited form as: Nat Struct Mol Biol. 2011 Dec 4;19(1):17–24. doi: 10.1038/nsmb.2177

Figure 3.

Figure 3

RAD51 is required for origin independent fork restart and reloading of replisome components after fork collapse. In (a) and (b) replication fork restart was monitored following incubation of sperm nuclei in the 1st extract for 60 min with or without 10 μg/ml aphidicolin and then transferring nuclear fractions that were untreated or briefly incubated with Mung bean nuclease to a 2nd extract containing 320 nM geminin, 1 mM roscovitine and GST or GST-BRC4 (a), or to mock or RAD51-depleted extracts containing 25, 50, 100 nM recombinant RAD51 (b). Replication products were monitored by incorporation of 32P-dATP added to the 2nd extract and resolved by alkaline (a) or neutral agarose gel (b) and subjected to autoradiography. Quantification of signals is shown at the bottom of the gel in (a) and in the graph (b). In (c) chromatin binding of RAD51 and CDC45 was monitored in egg extracts that were mock or RAD51 depleted and supplemented with the indicated amount of recombinant RAD51 (rRAD51). The status of replication fork proteins bound to chromatin isolated from extracts treated as in (a) is shown in (d).