A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

Background: Recently, we have discovered site-specific endonucleases, which recognize and cleave only DNA sequences with 5-methylcytosine. Two specificities of such endonucleases have been described. Enzymes BisI, BlsI, and GluI are isoschizomers and hydrolyze the DNA sequence 5'-GCNGC-3'/3'-CGNCG-5', which is methylated in different ways. The enzyme GlaI cleaves the DNA sequence 5'-GCGC-3'/3'-CGCG-5' if there are two, three or four 5-methylcytosines. The goal of the present work is to study in detail the composition of recognition sequence and effect of the methylated cytosines on the efficiency of DNA cleavage by the methyl-directed DNA endonuclease GlaI

The methylation-dependent restriction endonuclease (REase) BisI (Gm5C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of m5C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four m5C in the two strands, or hemi-methylated sites containing two m5C in one strand; Group II enzymes only cut GCNGC sites containing three to four m5C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four m5C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.

BackgroundPreviously, we developed a simple method for carrying out a restriction enzyme analysis of eukaryotic DNA in silico, based on the known DNA sequences of the genomes. This method allows the user to calculate lengths of all DNA fragments that are formed after a whole genome is digested at the theoretical recognition sites of a given restriction enzyme. A comparison of the observed peaks in distribution diagrams with the results from DNA cleavage using several restriction enzymes performed in vitro have shown good correspondence between the theoretical and experimental data in several cases. Here, we applied this approach to the annotated genome of Drosophila virilis which is extremely rich in various repeats.ResultsHere we explored the combined approach to perform the restriction analysis of D. virilis DNA. This approach enabled to reveal three abundant medium-sized tandem repeats within the D. virilis genome. While the 225 bp repeats were revealed previously in intergenic non-transcribed spacers between ribosomal genes of D. virilis, two other families comprised of 154 bp and 172 bp repeats were not described. Tandem Repeats Finder search demonstrated that 154 bp and 172 bp units are organized in multiple clusters in the genome of D. virilis. Characteristically, only 154 bp repeats derived from Helitron transposon are transcribed.ConclusionUsing in silico digestion in combination with conventional restriction analysis and sequencing of repeated DNA fragments enabled us to isolate and characterize three highly abundant families of medium-sized repeats present in the D. virilis genome. These repeats comprise a significant portion of the genome and may have important roles in genome function and structural integrity. Therefore, we demonstrated an approach which makes possible to investigate in detail the gross arrangement and expression of medium-sized repeats basing on sequencing data even in the case of incompletely assembled and/or annotated genomes.

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