p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition

Cell Cycle. Nov-Dec 2003;2(6):596-603.

Abstract

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A 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. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/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 (p21PCNA-) or CDK (p21CDK-) 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 p21CDK-HA, but not p21PCNA-, 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 G1/S phase transition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CDC2-CDC28 Kinases / metabolism
  • Cyclin A / genetics
  • Cyclin A / metabolism
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / genetics
  • Cyclins / metabolism*
  • DNA Polymerase III / metabolism*
  • DNA Replication*
  • G1 Phase / physiology*
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Luminescent Proteins / metabolism
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • S Phase / physiology*

Substances

  • CDKN1A protein, human
  • Cyclin A
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Luminescent Proteins
  • Proliferating Cell Nuclear Antigen
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • DNA Polymerase III