Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells.
The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells. The altered polypeptide migrated faster in SDS-polyacrylamide gel as compared to the wild-type FV3 26K protein. Five spontaneous revertants derived from the mutant regained the migrational characteristic of the wild-type 26K protein, DNA-mt activity, and methylation of their DNA. We further show that the 26K polypeptide is a DNA-binding protein and that 80% of the enzyme activity can be eluted from an ssDNA affinity column. Taken together, these data support the conclusion that the 26K polypeptide is associated with DNA-mt activity.
D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D002460:Cell Line; D002846:Chromatography, Affinity; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005814:Genes, Viral; D007497:Iridoviridae / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D008745:Methylation; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010455:Peptides; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine