Biology of DNA restriction.

DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.

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Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

https://mmbr.asm.org/content/mmbr/47/3/345.full.pdf

Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts.

All organisms need to continuously adapt to changes in their environment. Through horizontal gene transfer, bacteria and archaea can rapidly acquire new traits that may contribute to their survival. However, because new DNA may also cause damage, removal of imported DNA and protection against selfish invading DNA elements are also important. Hence, there should be a delicate balance between DNA uptake and DNA degradation. Here, we describe prokaryotic antiviral defense systems, such as receptor masking or mutagenesis, blocking of phage DNA injection, restriction/modification, and abortive infection. The main focus of this review is on CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated), a prokaryotic adaptive immune system. Since its recent discovery, our biochemical understanding of this defense system has made a major leap forward. Three highly diverse CRISPR/Cas types exist that display major structural and functional differences in their mode of generating resistance against invading nucleic acids. Because several excellent recent reviews cover all CRISPR subtypes, we mainly focus on a detailed description of the type I-E CRISPR/Cas system of the model bacterium Escherichia coli K12.

The CRISPRs, They Are A-Changin': How Prokaryotes Generate Adaptive Immunity

P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

Jakob Reiser

Papers

Methylation and cleavage sequences of the EcoP1 restriction-modification enzyme☆

Steps in the reaction mechanism of the Escherichia coli plasmid P15-specific restriction endonuclease.

A simplified assay for endonuclease and ligase activities