Chromatin proteome of S-phase in IgG- and DONSON-depleted egg extract

The aim of the project was to analyse the role of DONSON during DNA replication reaction in Xenopus laevis egg extract. To test what is the impact of DONSON on replication we decided to remove DONSON from egg extract by immunodepletion and analyse its impact on DNA replication reaction. In the parallel experiments we have shown that without DONSON egg extract cannot synthesise nascent DNA. Here we wanted to examine the change of replicating chromatin proteome in absence of DONSON. Interphase Xenopus laevis egg extract was immunodepleted with control nonspecific IgG antibodies beads or DONSON-antibodies. The efficiency of immunodepletion was shown to be over 95%. DNA replication was set up in both IgG- and DONSON-depleted egg extracts and chromatin isolated in the middle of S-phase, when replication forks where abundant in chromatin fraction in IgG-depleted extract.