Use of peptides from p21 (Waf1/Cip1) to investigate PCNA function in Xenopus egg extracts.
Exp Cell Res
; 265(2): 242-51, 2001 May 01.
Article
en En
| MEDLINE
| ID: mdl-11302689
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.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Cromatina
/
Ciclinas
/
Antígeno Nuclear de Célula en Proliferación
/
Replicación del ADN
/
Inhibidores Enzimáticos
Límite:
Animals
/
Female
/
Humans
/
Male
Idioma:
En
Revista:
Exp Cell Res
Año:
2001
Tipo del documento:
Article
País de afiliación:
Reino Unido
Pais de publicación:
Estados Unidos