Restriction alleviation and modification enhancement by the ral gene of bacteriophage [lamda]

Bacteriophages have a variety of mechanisms to escape host-encoded restriction and modification (R-M) systems. Bacteriophage X has a gene, nil, whose product alleviates restriction and enhances modification by a host encoding a type IA R-M system. The genes of the type IA R-M systems encode an hetero-oligomeric DNA methyltransferase (MTase) that together with an additional subunit forms a DNA endonuclease (ENase). Both enzymatic activities share a common subunit for targetsite recognition; unmodified targets are normally a good substrate for the ENase, whereas hemimethylated targets, those that are modified on only one strand of the duplex, are a good substrate for the MTase. The MTase component of the type-IA R-M systems are the only prokaryotic MTases shown to have a strong preference for hemimethylated DNA in vivo, and are also the only systems in which restriction is alleviated by Ral. This thesis describes an investigation of the mechanism of Ral activity, both in vivo and in vitro. Unmodified Xrat phages are restricted by bacteria expressing type IA R-M systems, presumably restriction occurs before the infecting phage has time to expres's the ral gene. In order to examine Ral activity in vivo, the degree of modification of progeny phages was measured after a single round of infection on a restriction-proficient strain, and the prediction that the progeny of nzr phages are more likely to become modified than rat phages was confirmed. This modification would be advantageous to those phages that subsequently infect bacteria with an R-M system of the same specificity. Interestingly, in some hosts progeny phages were similarly modified irrespective of their nil genotype. Some strains of E. coli contain a defective prophage, termed Rac, that is known to encode a Ral-like antirestriction function, Lar. The inability to detect Ral activity was found to correlate with the presence of the Rac prophage; presumably lar is expressed in a sub-population of Rack hosts. The gene encoding lar was isolated by cloning DNA fragments from the Rac prophage in a plasmid vector and screening for anti-restriction activity. In this way, lar was localised to a 500 bp DNA fragment, and was further defined by site-directed mutagenesis in conjunction with an analysis of the phenotypes of the mutants, and the polypeptides produced. The predicted amino acid sequences of Ral and Lar are remarkably dissimilar, although a number of residues do align which may indicate important features for structure and/or function. The i-al gene has been overexpressed and its product purified. Methylation assays, with purified Ral and the type IA EcoKI MTase failed to show any change in methylation activity in the presence of Ral. There are many reasons why no effect of Ral was detected, not least that the purification process may disrupt the polypeptides. In order to overcome these problems, similar experiments were performed using extracts of cells overexpressing ml and the genes encoding the EcoKJ MTase. These extracts, like extracts from cells producing only the MTase, methylated synthetic hemimethylated substrates efficiently, but only methylated unmodified substrates poorly. This is inconsistent with the hypothesis that Ral changes the activity of EcoKI from a maintenance MTase to a de novo MTase. An alternative explanation, consistent with present results and observations, is that Ral both blocks ENase activity and stimulates MTase activity.