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 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 effects 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 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 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 experimental system to analyze the dependence of postreplicative processes on PCNA function. We have shown that successful chromatin assembly is specifically dependent on PCNA. However, systems for analyzing the far more complex mechanisms required for the replication of nuclear double-stranded DNA have proved so far to be refractory to specific PCNA depletion.
Mattock
H
H
Department of Surgery and Molecular Oncology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom.
Jares
P
P
Zheleva
D I
DI
Lane
D P
DP
Warbrick
E
E
Blow
J J
JJ
D000818:Animals; D000962:Antimalarials / Q000494:pharmacology; D015153:Blotting, Western; D002738:Chloroquine / Q000494:pharmacology; D002843:Chromatin / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects* / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000494:pharmacology / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology; D014020:Tissue Extracts / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D000962:Antimalarials; C493956:CDKN1A protein, human; D002843:Chromatin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D010455:Peptides; D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D014020:Tissue Extracts; D002738:Chloroquine; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase