The regulation of genes governing restriction enzymes, including both DNA methylases and endonucleases, will be examined in Streptococcus pneumoniae. Also, the structures of these enzymes and their interactions with methylated and unmethylated DNA will be determined. Different strains of S. pneumoniae have different patterns of DNA methylation and harbor one of two complementary restriction enzyme systems. Strains that contain the methylated sequence 5'-GmeATC-3' produce DpnII, which cleaves 5'-GATC-3', and two methylases, DpnM and DpnA. Strains not methylated in this sequence produce DpnI, which is an unusual restriction endonuclease in that it cleaves only the methylated sequence, 5'-GmeATC-3' The genes encoding the two restriction systems are located on cassettes alternatively situated at the same point in the bacterial chromosome. The cassettes have been cloned, transferred, and expressed in strains of both S. pneumoniae and Escherichia coli. Two regulatory phenomena of restriction gene expression will be explored. One allows the transfer of the DpnlI system by expressing the methylase prior to the endonuclease. The other allows rare, direct transitions between DpnI and DpnII systems. The regulatory mechanisms will be investigated by identifying and characterizing products of transcription and of mRNA processing and by analyzing the effect on transcription of DNA methylation. Putative atypical ribosome binding sites, different from Shine-Dalgarno sequences, were identified in the DpnII system; their role in ribosome binding and gene expression will be assessed. Possible activities of a protein of unknown function produced by the DpnI system will be explored. On the structural side, the role in binding to methylated DNA of a zinc-finger motif in the DpnI endonuclease protein will be examined. Three DpnII system proteins that all recognize 5'-GATC-3', but have different primary sequences, will be subjected to x-ray crystallography in the presence and absence of bound DNA substrate to determine their structures and their modes of interaction with the recognition site in DNA. These studies should further our understanding of genetic mechanisms significant to pathogenesis in S. pneumoniae, of the regulation of gene expression by DNA methylation and other mechanisms, and of the molecular bases of DNA-protein interaction, all of which have ramifications with respect to human health.
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