Use of Peptides from p21 (Waf1/Cip1) to Investigate PCNA Function in Xenopus Egg Extracts Heidi Mattock, Pedro Jares,* Daniella I. Zheleva,† David P. Lane, Emma Warbrick, 1 and J. Julian Blow* Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom; *Department of Biochemistry, University of Dundee, MSI/WTB Complex, Dundee DD1 5EH, United Kingdom; and †Cyclacel Limited, Dundee Technopole, James Lindsay Place, Dundee DD1 SJJ United Kingdom Cell-free systems derived from unfertilized Xenopus eggs have been particularly informative in the study of the regulation and biochemistry of DNA replication. We have developed a Xenopus-based system to analyze proliferating cell nuclear antigen (PCNA)-specific ef- fects on the functional properties of egg extracts. To do this, we have coupled peptides derived from p21 (Waf1/Cip1) to beads and used these to deplete PCNA from Xenopus egg extracts. The effect on various as- pects 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 upon PCNA. By adding exogenous T7 DNA polymerase to PCNA- depleted extracts, we have uncoupled processive DNA replication from PCNA activity and so created an ex- perimental system to analyze the dependence of post- replicative processes on PCNA function. We have shown that successful chromatin assembly is specifi- cally dependent on PCNA. However, systems for ana- lyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA deple- tion. © 2001 Academic Press Key Words: p21(Waf1/Cip1); PCNA; cyclin-dependent kinase; DNA replication; chromatin assembly. INTRODUCTION p21, also known as Waf1, Cip1, etc., is a small reg- ulatory protein that contains several functional do- mains, including those for interactions with Cdks, cy- clins, and proliferating cell nuclear antigen (PCNA). p21 plays an important role in the coordination of the cellular pathways that act in response to DNA damage to repair DNA lesions. The mechanism of p21 function is still not clear, but it appears to differentially regu- late specific Cdk– cyclin complexes. For example, re- cent evidence has shown that although p21 acts as a potent inhibitor of cyclin A–Cdk2, it does not efficiently inhibit cyclin D–Cdk4 activity and may positively reg- ulate such complexes by acting as an assembly factor [1– 4]. There is good evidence to indicate that p21 can in- hibit DNA replication via a mechanism that is depen- dent on PCNA, though the physiological role of the p21–PCNA interaction is not fully understood [5– 8]. The site of p21 interaction on PCNA is shared by many proteins involved in the mechanics of DNA replication [9], and it is possible that p21 inhibits progressive DNA replication by competitively blocking the interactions of such proteins. Analysis of the interaction between human p21 and PCNA resulted in the identification of a small 20- amino-acid peptide derived from the C terminus of p21 that was sufficient to interact with PCNA and was also capable of inhibiting SV40 DNA replication in vitro [10]. Further studies have shown that either full- length p21 or p21-derived regions can inhibit DNA replication in a PCNA-dependent manner, and al- though p21 does not appear to inhibit DNA repair, a synthetic PCNA-binding p21 peptide can affect nucle- otide excision repair in vitro [5– 8, 11]. In vitro studies have shown that the C-terminal region of p21, which contains the PCNA-interacting domain, can competi- tively block reassociation of the polymerase complex with PCNA. Although this does not significantly affect short patches of repair synthesis, the long tract DNA synthesis necessary for replication is prevented [11, 12]. Observations that the formation of PCNA–p21 complexes is part of the cellular response to DNA dam- age underline the physiological significance of the p21– PCNA interaction [13]. However, although it was pre- viously thought that the Cdk– cyclin inhibitory activity of p21 resided solely in an N-terminal domain, it is now recognized that a secondary inhibitory motif is located adjacent to the PCNA-interaction domain at the C terminus [14, 15]. This means that results obtained using small C-terminal regions of p21 are not neces- sarily PCNA-dependent effects. In the accompanying paper [15a], we describe the effects of expressing small 1 To whom reprint requests should be addressed. Fax: (01382) 496 363. E-mail: e.warbrick@dundee.ac.uk. 242 0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Experimental Cell Research 265, 242–251 (2001) doi:10.1006/excr.2001.5181, available online at http://www.idealibrary.com on