Interaction of CR6 (GADD45gamma ) with proliferating cell nuclear antigen impedes negative growth control.

GADD45, MyD118, and CR6 (also termed GADD45alpha, beta, and gamma) 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.

Human DNA-demethylating activity: a glycosylase associated with RNA and PCNA.

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.

Characterization of MyD118, Gadd45, and proliferating cell nuclear antigen (PCNA) interacting domains. PCNA impedes MyD118 AND Gadd45-mediated negative growth control.

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.

Partial purification and characterization of human 5-methylcytosine-DNA glycosylase.

The molecular mechanisms by which DNA 5-methylcytosine content is modulated are incompletely understood. Reduction of DNA 5-methylcytosine content has been correlated with the transition from hyperplasia to adenoma in the genesis of human adenocarcinoma of the colon. 5-methylcytosine-DNA glycosylase removes 5-methylcytosine from DNA as a free base, but its involvement in this process is unknown. The 5-methylcytosine-DNA glycosylase activity in HeLa nuclear extracts has been partially purified, with a 460-fold enrichment, and characterized. This activity is specific for 5-methylcytosine at CpG sites in fully methylated DNA; hemimethylated DNA is not a significant substrate. DNA containing unmethylated cytosines is not cleaved by the enzyme. There is an absolute requirement for Mg2+ ions for the activity, which is inhibited by EDTA. This 5-methylcytosine-DNA glycosylase activity could be involved in carcinogenesis, transcription, replication, differentiation and development through resultant DNA hypomethylation following enzymatic removal of 5-methylcytosine from DNA.

The differentiation primary response gene MyD118, related to GADD45, encodes for a nuclear protein which interacts with PCNA and p21WAF1/CIP1.

Towards dissecting the regulation of terminal differentiation, including growth arrest and apoptosis, myeloid differentiation primary response (MyD) genes, induced in the absence of de novo protein synthesis following induction of M1 myeloblastic leukemia cells for terminal differentiation have been isolated. MyD118 was one of the novel MyD genes cloned, subsequently observed also to be a primary response gene to TGF-beta, which induces M1 cells for growth arrest and apoptosis uncoupled from differentiation. The MyD118 encoded protein was observed to be remarkably similar to the protein encoded by Gadd45, a growth arrest and DNA damage induced gene, regulated in part by the tumor suppressor p53. Though evidence has accumulated that MyD118 functions as an important modulator of negative growth control both in hematopoietic and non-hematopoietic cells, its mechanism of action is unknown. To better understand the role(s) of MyD118 in negative growth control, we have analysed the expression and biological characteristics of the MyD118 protein, compared to the Gadd45 protein, in distinct pathways of growth arrest and apoptosis, including p53 dependent and independent pathways either coupled or uncoupled from differentiation. It is shown that MyD118 and Gadd45 differentially accumulated upon induction of distinct pathways of growth arrest and apoptosis; notably, MyD118, but not Gadd45, was induced by TGF-beta, whereas Gadd45, but not MyD118, was induced by activating wild type (wt) p53 function. It is also shown that MyD118 is a nuclear protein, which regardless of the pathway induced, predominantly localized within the cell nucleus, and interacted with the DNA replication and repair protein PCNA and the cyclin dependent kinase inhibitor P21WAF1/CIP1. MyD118 also modestly stimulated DNA repair in vitro. All of these characteristics were shared with Gadd45. Finally, it is demonstrated that MyD118, Gadd45 and p21 synergized in the suppression of colony formation by NIH3T3 cells. Taken together, these findings demonstrate that MyD118 and Gadd45 are representative of a new protein family that share remarkable functional similarities in the control of distinct pathways of negative growth, including the suppression of cellular growth and programmed cell death.

Synthesis and properties of DNA purine dehydrodimers: 8-8-bideoxyribonucleosides and 8-8-bideoxyribonucleotides.

Purine dimers are formed by oxidation of DNA. There is evidence that these dimers are not repaired by cells from the human disease xeroderma pigmentosum. It has been suggested that unrepaired purine dimers are involved in the etiogenesis of internal cancers and neural degeneration that are observed in this disease. In order to study the properties and biological consequences of such moieties, these compounds were synthesized: 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine-5'-monophosphate; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine, and 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine-5'-monophosphate. Following purification, they were characterized by mass spectrometry and nuclear magnetic resonance studies. Ultraviolet, fluorescence, and circular dichroic spectra of these products were established. The behavior of these photoproducts in various chromatographic systems was elucidated. Syntheses of purine dimers and descriptions of their properties can aid the studies of their possible formation in, and excision from, oxidized DNA.

Excision of ultraviolet-induced photoproducts of 5-methylcytosine from DNA.

Transition mutations at DNA 5-methylcytosines, congregated at CpG islands, are implicated in the etiogenesis of human diseases. Formation of 5-methylcytosine hydrate (5-methyl-6-hydroxy-5,6-dihydrocytosine) by hydration of the 5,6 double bond of 5-methylcytosine has been suggested as an intermediate in a possible mechanism of deamination to thymine. Ultraviolet irradiation of DNA yields pyrimidine hydrates, which are removed by repair glycosylases. We have identified 5-methylcytosine photoproducts following their excision from DNA by E. coli endonuclease III. Poly(dG-[3H]5-medC):poly(dG-[3H]5-medC) was irradiated and reacted with the enzyme. Radiolabeled photoproduct releases were directly proportional to irradiation doses and enzyme concentrations. These were identified as cis-thymine hydrate (6-hydroxy-5,6-dihydrothymine) and trans-thymine hydrate. Recovery of thymine hydrates is consistent with hydration of pyrimidines. Subsequent heating (which converts thymine hydrates to thymines) and chemical sequencing of an irradiated, 3' end-labeled, synthetic DNA strand demonstrated the appearance of thymine at the 5-methylcytosine site. These results demonstrate a mechanism for deamination of DNA 5-methylcytosine via hydration of the 5,6 double bond, putatively yielding 5-methylcytosine hydrate; this deaminates to thymine hydrate, and loss of water yields thymine formation at the 5-methylcytosine site. Identification of these DNA 5-methylcytosine modified moieties indicates a possible molecular mechanism for the frequent transition mutations found at CpG loci.

Enzymic removal of 5-methylcytosine from DNA by a human DNA-glycosylase.

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.