Hashimoto Y, Puddu F, Costanzo V.

Cancer Research UK, 206281 European Research Council, A15668 Cancer Research UK, A7124 Cancer Research UK, CRUK_A15668 Cancer Research UK, CRUK_A7124 Cancer Research UK, ERC_206281 European Research Council

	Figure 2. RAD51 is required for stable chromatin association of fork proteins in the presence template breakage. In (a) fork proteins association to chromatin isolated from extracts treated with GST or GST-BRC4 and 0, 2.92, 0.97, and 0.32 U μl−1 S1 nuclease in the presence of 1μg ml−1 aphidicolin (Low aph) was analysed. In (b) we monitored the chromatin status of fork proteins, histone H2AX and PCNA in extracts treated with GST or GST-BRC4 2.92 (1/100) and 0.37 (1/800) U μl−1 S1 nuclease and aphidicolin. In (c) chromatin binding of PSF2 and CDC45 in the presence of 0.97 U μl−1 S1 nuclease was analysed in mock or RAD51 depleted extracts. In (d) chromatin binding of the indicated proteins over time in extracts treated with GST or GST-BRC4 and 1.46 U μl−1 of S1 nuclease and aphidicolin was monitored. In (e) nuclear Chk1 phosphorylation on Ser 345 was monitored in extracts treated with 1μg/ml aphidicolin alone or in combination with 1.46 U μl−1 of S1 nuclease. W.B in a–e were performed using antibodies against the indicated chromatin binding factors. Ext: 0.5 μl egg extract was loaded as control in (b) and (d). Chromatin and nuclear fractions were isolated 60 min after the addition of sperm DNA to egg extracts unless otherwise indicated.
	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).
	Figure 4. MRE11 nuclease activity is required for DNA replication upon fork collapse. In (a) and (b) the effects of MRE11 nuclease inhibitor mirin on replication of sperm nuclei that were untreated or treated with MMS in the presence or absence of GST-BRC4 (a) or on sperm nuclei incubated in extracts treated with 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and aphidicolin were monitored (b). Replication products were monitored by 32P-dATP incorporation and resolved by neutral agarose gels, which were subjected to autoradiography. Signal intensities were reported in the graphs. In (c) the effect of mirin on replication proteins bound to chromatin isolated after 50 min incubation in extracts treated with 0, 1.46, 0.73, 0.37 and 0.18 U μl−1 S1 nuclease was analysed. In (d) the binding of the indicated fork proteins to chromatin incubated for 45 min in egg extracts that were untreated or supplemented with 0.73 U μl−1 S1 nuclease and mirin was monitored following protein crosslinking, sonication induced DNA fragmentation and immunoprecipitation with control and anti-CDC45 serum. * non-specific band. Ext: 0.5 μl egg extract was loaded as a control in (c) and (d).
	Figure 5. The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In (a) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). (b) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or 0.1 U μl−1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). (c) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.
	Figure 6. A model of replication fork collapse and restart: The presence of a ssDNA lesion in the template creates one-sided DSB at the passage of the replisome (1) leading to the dissociation of the GINS and Pol epsilon from the fork, whereas MCM and CDC45 remain stably bound to collapsed forks (2). The one-sided DSB undergoes MRE11 mediated nuclease resection and RAD51 dependent strand annealing/invasion of the intact template. The MRE11 complex might also tether the broken DNA strand to the intact one (3). This process requires BIR proficient PCNA, which promotes Pol eta dependent strand extension (4). Reloading of the GINS and Pol epsilon in an origin independent fashion promotes reassembly of functional replisome (5).

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