Characterization of mutations of the bacteriophage P1 mod gene encoding the recognition subunit of the EcoP1 restriction and modification system

Abstract

This study characterized several mutations of the bacteriophage P1 mod gene. This gene codes for the subunit of the EcoP1 restriction enzyme that is responsible for DNA sequence recognition and for modification methylation. We cloned the mutant mod genes into expression vectors and purified the mutant proteins to near homogeneity. Two of the mutant mod genes studied were the c2 clear-plaque mutants described by Scott (Virology 41:66-71, 1970 Evidence is presented that one of them, Ser-240 -* Pro, simultaneously lost the capacity to bind SAM and may also have changed its DNA sequence specificity.The prophage P1 and the related plasmid P15B both code for DNA restriction and modification systems: EcoPl and EcoP15, respectively. While these systems recognize different DNA sequences (AGACC for EcoPl and CAGCAG for EcoP15 [5,14]), they are biochemically and physiologically very similar (2, 13) and are sufficiently different from other DNA restriction and modification systems that, together with a system coded in some strains of Haemophilus influenzae, they are considered to form a functionally distinct group that has been called type III (20). Type III restriction enzymes contain two different subunits, the products of the res and mod genes (13, 18). The enzymes have an absolute requirement for ATP, although they do not hydrolyze it, and they are stimulated by S-adenosylmethionine (SAM), a compound which is also the methyl donor in the modification reaction. The restriction enzymes are multifunctional and catalyze both restriction and modification. In the presence of both ATP and SAM, restriction and modification are competing reactiohs and only partial digests are obtained (20,27). Although type III restriction enzymes have modification activity, separate modification methylases can also be isolated. These are composed of the mod gene product only, and methylation is their only detectable enzymatic activity (13).Genetic analysis of the EcoPl and EcoPl5 systems showed that two contiguous genes were involved. Transposon mutagenesis showed that one of them, called res, was required for restriction only, while the other, called mod, was necessary for both restriction and modification (6, 18). The res gene product has a molecular weight of 106,000, and DNA heteroduplex analysis indicates that the EcoPl and EcoP15 res genes are virtually identical (18). The mod gene product has a molecular weight of about 75,000. A compar-* Corresponding author. t Permanent address:

Biology of DNA restriction.

“…Both c2 mutant proteins were purified. As expected from the in vivo phenotype, they had no methylase activity and their primary defect was that they failed to bind the methyl donor, AdoMet (162). gene have also been constructed, and they encode mutants with phenotypes similar to those of the c2 mutants (162).…”

“…As expected from the in vivo phenotype, they had no methylase activity and their primary defect was that they failed to bind the methyl donor, AdoMet (162). gene have also been constructed, and they encode mutants with phenotypes similar to those of the c2 mutants (162). One of these mutants also had the interesting property that, unlike the wild-type enzyme, it was no longer subject to inhibition by high concentrations of AdoMet (163).…”

In vivo genetic exchange of a functional domain from a type II A methylase between lactococcal plasmid pTR2030 and a virulent bacteriophage

“…Other antirestriction mechanisms include modification other than methylation (e.g., glycosylation or the introduction of unusual bases) and selective reduction or elimination of the specific target sequences recognized by the host endonucleases (21,28). In E. coli and Bacillus sp., a number of temperate bacteriophages have been identified which encode methylases that modify the host endonuclease target sequences (20,22,33 convergent evolution of function. A common, though distant, evolutionary origin has been suggested for the phage T4 dam and host dam genes, even though there is a complete lack of DNA-DNA homology (6).…”

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox

“…We were unable to detect endonuclease activity in cell-free extracts prepared from M. xanthus DK883, the original source of Mx8, or from extracts prepared from a lysogen of host DZ1 carrying wild-type Mx8 as prophage with DNA prepared from Mx8 del1 or coliphage particles as substrates. Furthermore, we were unable to detect endonuclease activity in extracts prepared from a second nat- (16,19). In contrast, phage Mx8 del1 forms turbid plaques and stable lysogens of both DZ1 and DK6204 (data not shown).…”

“…To understand why myxophage Mx8 encodes a site-specific methylase, we explored four potential roles of this methylase in the physiology of the phage. First, because DNA methylases are often genetically linked with their cognate restriction endonucleases, we asked whether Mox might be a part of a phage-encoded restriction-modification system, like the coliphage P1-encoded Mod methylase (16,19). We were unable to detect endonuclease activity in cell-free extracts prepared from M. xanthus DK883, the original source of Mx8, or from extracts prepared from a lysogen of host DZ1 carrying wild-type Mx8 as prophage with DNA prepared from Mx8 del1 or coliphage particles as substrates.…”