Gadd45a (Gadd45), Gadd45b (MyD118), and Gadd45g (CR6) constitute a family of evolutionarily conserved, small, acidic, nuclear proteins, which have been implicated in terminal differentiation, growth suppression, and apoptosis. How Gadd45 proteins function in negative growth control is not fully understood. Recent evidence has implicated Gadd45a in inhibition of cdc2/cyclinB1 kinase and in G2/M cell cycle arrest. Yet, whether Gadd45b and/or Gadd45g function as inhibitors of cdc2/cyclinB1 kinase and/or play a role in G2/M cell cycle arrest has not been fully established. In this work, we show that Gadd45b and Gadd45g specifically interact with the Cdk1/CyclinB1 complex, but not with other Cdk/Cyclin complexes, in vitro and in vivo. Data also has been obtained that Gadd45b and Gadd45g, as well as GADD45a, interact with both Cdk1 and cyclinB1, resulting in inhibition of the kinase activity of the Cdk1/cyclinB1 complex. Inhibition of Cdk1/cyclinB1 kinase activity by Gadd45b and Gadd45a was found to involve disruption of the complex, whereas Gadd45g did not disrupt the complex. Moreover, using RKO lung carcinoma cell lines, which express antisense Gadd45 RNA, data has been obtained, which indicates that all three Gadd45 proteins are likely to cooperate in activation of S and G2/M checkpoints following exposure of cells to UV irradiation.

MyD118 and Gadd45 are related genes encoding for proteins that play important roles in negative growth control, including growth suppression and apoptosis. MyD118 and Gadd45 are related proteins that previously were shown to interact with proliferating cell nuclear antigen (PCNA), implicated in DNA replication, DNA repair, and cell cycle progression. To establish the role of MyD118 and Gadd45 interactions with PCNA, in this work we sought to identify the interacting domains and analyze the significance of this interaction in negative growth control. Using complementary in vivo and in vitro interaction assays the N-terminal (1-46) and middle (100 -127) regions of PCNA were identified as harboring MyD118-and Gadd45 interacting domains, whereas PCNA interacting domains within MyD118 and Gadd45 were localized to the C termini of these proteins (amino acids 114 -156 and 137-165, respectively). These findings provide first evidence that similar domains within MyD118 and Gadd45 mediate interactions with PCNA. Importantly, ectopic expression of MyD118 or Gadd45 N-terminal peptides, lacking the PCNA interacting domain, was found to suppress colony formation or induce apoptosis more efficiently than the full-length proteins. These findings suggest that interaction of MyD118 or Gadd45 with PCNA, in essence, serves to impede negative growth control.

GADD45, MyD118, and CR6 (also termed GADD45␣, ␤, and ␥) comprise a family of genes that encode for related proteins playing important roles in negative growth control, including growth suppression. Data accumulated suggest that MyD118/GADD45/CR6 serve similar but not identical functions along different apoptotic and growth suppressive pathways. It is also apparent that individual members of the MyD118/GADD45/CR6 family are differentially induced by a variety of genetic and environmental stress agents. The MyD118, CR6, and GADD45 proteins were shown to predominantly localize within the cell nucleus. Recently, we have shown that both MyD118 and GADD45 interact with proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA replication, DNA repair, and cell cycle progression, as well as with the universal cyclin-dependent kinase inhibitor p21. In this work we show that also CR6 interacts with PCNA and p21. Moreover, it is shown that CR6 interacts with PCNA via a domain that also mediates interaction of both GADD45 and MyD118 with PCNA. Importantly, evidence has been obtained that interaction of CR6 with PCNA impedes the function of this protein in negative growth control, similar to observations reported for MyD118 and GADD45 (1).

DNA 5-methylcytosine is a major factor in the silencing of mammalian genes; it is involved in gene expression, differentiation, embryogenesis and neoplastic transformation. A decrease in DNA 5-methylcytosine content is associated with activation of specific genes. There is much evidence indicating this to be an enzymic process, with replacement of 5-methylcytosine by cytosine. We demonstrate here enzymic release of 5-methylcytosines from DNA by a human 5-methylcytosine-DNA glycosylase activity, which affords a possible mechanism for such replacement. This activity generates promutagenic apyrimidinic sites, which can be related to the high frequency of mutations found at DNA 5-methylcytosine loci. The recovery of most released pyrimidines as thymines indicates subsequent deamination of free 5-methylcytosines by a 5-methylcytosine deaminase activity. This prevents possible recycling of 5-methylcytosine into replicative DNA synthesis via a possible 5-methyl-dCTP intermediate synthesized through the pyrimidine salvage pathway. Taken together, these findings indicate mechanisms for removal of 5-methylcytosines from DNA, hypermutability of DNA 5-methylcytosine sites, and exclusion of 5-methylcytosines from DNA during replication.

We have partially purified and characterized the 5-methylcytosine removing activity (5-meC-DNA Glycosylase) from HeLa cells with 700-fold enrichment. This activity cleaves DNA specifically at fully methylated CpG sites. The mechanism of 5-meC removal is base excision from fully methylated CpG loci on DNA, producing abasic sites. Hemi-methylated DNA is not a substrate. A prominent 52 KDa protein is present in all partially purified fractions. This activity is tightly associated with other nuclear factors and proteins, which resulted in differential fractionation of this activity on ion exchange columns. One nuclear factor associated with this activity is identified as RNA. Another nuclear protein, proliferating cell nuclear antigen (PCNA) is also associated with this enzyme. Glycosylic removal of 5-meC from DNA by this activity could be involved in the regulation of transcription, replication, differentiation, and development through resultant hypomethylation of DNA.