Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts

Cell-free systems derived from unfertilized Xenopus eggs have been particul arly informative in the study of the regulation and biochemistry of DNA rep lication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific effects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf 1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various aspects of DNA replication can be analyzed after the readdition of PCNA and other purified proteins. Using this system, we have shown that replication of single-stranded M13 DNA is entirely dependent upo n PCNA. By adding exogenous T7 DNA polymerase 60 PCNA-depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so create d an experimental system to analyze the dependence of post-replicative proc esses on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far mor e complex mechanisms required for the replication of nuclear double-strande d DNA have proved so far to be refractory to specific PCNA depletion. (C) 2 001 Academic Press.