RESUMEN
Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction-modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo I/química , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Secuencia de Bases , ADN/química , Desoxirribonucleasas de Localización Especificada Tipo I/clasificaciónRESUMEN
Restriction and modification are two opposing activities that are used to protect bacteria from cellular invasion by DNA (e.g. bacteriophage infection). Restriction activity involves cleavage of the DNA; while modification activity is the mechanism used to "mark" host DNA and involves DNA methylation. The study of Type I restriction enzymes has often been seen as an esoteric exercise and this reflects some of their more unusual properties - non-stoichiometric (non-catalytic) cleavage of the DNA substrate, random cleavage of DNA, a massive ATPase activity, and the ability to both cleave DNA and methylate DNA. Yet these enzymes have been found in many bacteria and are very efficient as a means of protecting bacteria against bacteriophage infection, indicating they are successful enzymes. In this review, we summarise recent work on the mechanisms of action, describe switching of function and review their mechanism of action. We also discuss structural rearrangements and cellular localisation, which provide powerful mechanisms for controlling the enzyme activity. Finally, we speculate as to their involvement in recombination and discuss their relationship to helicase enzymes.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Bacterias/enzimología , Bacterias/genética , Metilación de ADN , Enzimas de Restricción-Modificación del ADN , ADN Bacteriano/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/clasificación , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Interacciones Huésped-Patógeno , Modelos Moleculares , Proteínas Motoras Moleculares/metabolismoRESUMEN
KpnBI is a restriction-modification (R-M) system recognized in the GM236 strain of Klebsiella pneumoniae. Here, the KpnBI modification genes were cloned into a plasmid using a modification expression screening method. The modification genes that consist of both hsdM (2631 bp) and hsdS (1344 bp) genes were identified on an 8.2 kb EcoRI chromosomal fragment. These two genes overlap by one base and share the same promoter located upstream of the hsdM gene. Using recently developed plasmid R-M tests and a computer program RM Search, the DNA recognition sequence for the KpnBI enzymes was identified as a new 8 nt sequence containing one degenerate base with a 6 nt spacer, CAAANNNNNNRTCA. From Dam methylation and HindIII sensitivity tests, the methylation loci were predicted to be the italicized third adenine in the 5' specific region and the adenine opposite the italicized thymine in the 3' specific region. Combined with previous sequence data for hsdR, we concluded that the KpnBI system is a typical type I R-M system. The deduced amino acid sequences of the three subunits of the KpnBI system show only limited homologies (25 to 33% identity) at best, to the four previously categorized type I families (IA, IB, IC, and ID). Furthermore, their identity scores to other uncharacterized putative genome type I sequences were 53% at maximum. Therefore, we propose that KpnBI is the prototype of a new 'type IE' family.
Asunto(s)
Enzimas de Restricción del ADN/clasificación , Desoxirribonucleasas de Localización Especificada Tipo I/clasificación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Metilación de ADN , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Enzimas de Restricción-Modificación del ADN/genética , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Prueba de Complementación Genética , Klebsiella pneumoniae/enzimología , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Restriction endonucleases have become a fundamental tool of molecular biology with many commercial vendors and extensive product lines. While a significant amount has been learned about restriction enzyme diversity, genomic organization, and mechanism, these continue to be active areas of research and assist in classification efforts. More recently, one focus has been their exquisite specificity for the proper recognition sequence and the lack of homology among enzymes recognizing the same DNA sequence. Some questions also remain regarding in vivo function. Site-directed mutagenesis and fusion proteins based on known endonucleases show promise for custom-designed cleavage. An understanding of the enzymes and their properties can improve their productive application by maintaining critical digest parameters and enhancing or avoiding alternative activities.
Asunto(s)
Enzimas de Restricción del ADN/química , Enzimas de Restricción del ADN/clasificación , Animales , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo I/química , Desoxirribonucleasas de Localización Especificada Tipo I/clasificación , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Desoxirribonucleasas de Localización Especificada Tipo I/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/clasificación , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo III/química , Desoxirribonucleasas de Localización Especificada Tipo III/clasificación , Desoxirribonucleasas de Localización Especificada Tipo III/genética , Desoxirribonucleasas de Localización Especificada Tipo III/metabolismo , Activación Enzimática , Humanos , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo I/clasificación , Escherichia coli/enzimología , Klebsiella pneumoniae/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Enzimas de Restricción-Modificación del ADN/genética , ADN Bacteriano/análisis , Desoxirribonucleasas de Localización Especificada Tipo I/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Subunidades de Proteína , Salmonella enterica/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Restriction enzymes are well known as reagents widely used by molecular biologists for genetic manipulation and analysis, but these reagents represent only one class (type II) of a wider range of enzymes that recognize specific nucleotide sequences in DNA molecules and detect the provenance of the DNA on the basis of specific modifications to their target sequence. Type I restriction and modification (R-M) systems are complex; a single multifunctional enzyme can respond to the modification state of its target sequence with the alternative activities of modification or restriction. In the absence of DNA modification, a type I R-M enzyme behaves like a molecular motor, translocating vast stretches of DNA towards itself before eventually breaking the DNA molecule. These sophisticated enzymes are the focus of this review, which will emphasize those aspects that give insights into more general problems of molecular and microbial biology. Current molecular experiments explore target recognition, intramolecular communication, and enzyme activities, including DNA translocation. Type I R-M systems are notable for their ability to evolve new specificities, even in laboratory cultures. This observation raises the important question of how bacteria protect their chromosomes from destruction by newly acquired restriction specifities. Recent experiments demonstrate proteolytic mechanisms by which cells avoid DNA breakage by a type I R-M system whenever their chromosomal DNA acquires unmodified target sequences. Finally, the review will reflect the present impact of genomic sequences on a field that has previously derived information almost exclusively from the analysis of bacteria commonly studied in the laboratory.