期刊
CELL CYCLE
卷 2, 期 6, 页码 596-603出版社
TAYLOR & FRANCIS INC
DOI: 10.4161/cc.2.6.502
关键词
p21(waf1/cip1); PCNA; DNA polymerase delta; DNA replication sites; green fluorescent protein; DNase I
类别
资金
- CNR Target Project Biotechnology
- La Ligue Nationale Contre le Cancer
The ability of the cyclin-dependent kinase (CDK) inhibitor p21(CDKN1A) to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G(1) phase cell cycle arrest induced by p21GFP occurred also at the G(1)/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21(PCNA-)) or CDK (p21(CDK-)) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21(CDK)-HA, but not p21(PCNA-), co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G(1)/S phase transition.
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