CHARACTERIZATION OF THE INTERACTION BETWEEN PCNA AND GADD45

We have examined the interaction between the DNA replication and repair protein PCNA, and the growth arrest and DNA damage induced protein Gadd45. An anti-Gadd45 polyclonal antibody co-immunoprecipitates PCNA but in reciprocal experiments, an anti-C terminal anti-PCNA antibody failed to co-immunoprecipitate Gadd45, We used a yeast two hybrid assay to demonstrate that human Gadd45 interacts with both human and S. pombe PCNA, We have determined that the N-terminal 94 amino acids of Gadd45 bind to PCNA, and using a series of N-terminal and C-terminal deletions of human PCNA we have mapped two potential Gadd45 binding sites, Deletion of the last 6 amino acids of PCNA ablated interaction, suggesting a role in Gadd45 binding, This explains the inability of an anti-C terminal PCNA antibody to co-immunoprecipitate Gadd45, Using a peptide ELISA approach, we showed that Gadd45 protein binds strongly to three regions of PCNA (residues 1-20, 61-80, and 196-215) and weakly to residues 121-170, The crystal structure of PCNA provides insight into our genetic and immunochemical data, Our results confirm an interaction between PCNA and Gadd45, define regions of both molecules involved in this interaction, and are consistent with a potential stoichiometry of 2 Gadd45 molecules to each PCNA monomer. These data provide support for the notion that PCNA-Gadd45 interactions co-ordinate cell cycle and DNA repair.