Figure 5: PTEN is physically associated with DNA replication forks and PTEN recruits Rad51 on chromatin for stalled fork recovery.

(a) Chromatin was isolated from PTEN^+/+ and PTEN^−/− DLD-1 cells in the presence and absence of 2 mM HU for indicated time periods, before PTEN immunoblotting. (b) Cell lysates were prepared from PTEN^+/+ DLD-1 cells following sonication-induced chromatin lysis. Cells with and without HU treatment (2 mM, 2 h) were subjected to PTEN immunoprecipitation followed by immunoblotting of Rad51 and Chk1. (c) PTEN^+/+ and PTEN^−/− HCT116 cells were labelled with EdU and treated with 2 mM HU. Nascent DNA was conjugated with biotin for iPOND analysis as described in Methods. DNA replication fork-associated PTEN, Rad51 and Chk1 were detected by western blotting. Histone H3 was used a loading control. (d) A schematic model to show formation of the PTEN-Rad51-Chk1 complex on DNA replication forks in normal conditions and Rad51 dissociation (Chk1 may retain) following PTEN depletion. (e) PTEN overexpression in Pten^−/− MEFs. (f,g) Pten^−/− MEFs containing ectopic PTEN were treated with different doses of HU, before sequential IdU-CldU pulse labelling. The frequency of stalled forks and fork restart (stalled and elongating type as depicted in Fig. 2g) was scored. Data are presented as means±s.e.m. and analysed with unpaired t-test. **P<0.01; ***P<0.001. (h) Pten^−/− MEFs with and without ectopic PTEN were subjected to isolation of chromatin fraction, followed by western analysis of Pten, Rad51 and Chk1. Ncl was used to indicate equal loading of chromatin fractions.