Biology of DNA Restriction

Restriction-Modification

• Phenomenon first described 40 yrs ago in viruses
• Restriction occurs by an endonucleolytic cleavage of foreign DNA
• 2 different types:
• one where only DNA that carries specific modifications is cleaved (host DNA avoids restriction because it is not modified)
• other where cellular DNA is protected from restriction by modification i.e. by methylation of adenosyl or cytosyl residues within the sequences recognized by Restriction Enzymes -- The classical R-M systems

Restriction-Modification (R-M) systems

Type I

• Most complex system
• Main enzyme
• 3 subunit protein containing the products of hsd = host specificity for DNA:
• hsds
• hsdm
• hsdr
• Multifunctional → catalyzes both restriction and modification reactions
• Is a restriction-dependent ATPase and a DNA topoisomerase
• Second enzyme
• hsds + hsdm
• Monofunctional → modification methylase
• Has no physiological role under normal conditions
• Type I systems have only been found so far in Enterobacteriaceae with the exception of H. influenzae
• Requires co-factors
• 3 families:
• Type IA
• Type IB → EcoAI, EcoEI, CfrII
• Type IC → StyR124I (Salmonella conjugative plasmid)

Type II

• Simplest R-M system
• Separate restriction and modification enzymes
• No special co-factor requirements except Mg2+
• Endonucleases and methyltransferases are products of independent evolution
• Expression is tightly regulated
• Examples:
• EcoRII
• requires at least 2 restriction sites for cleavage with maximum distance between them being 1Kb
• needs to bind to an activator site
• Type IIS
• recognize asymmetrical 4-7 bp DNA sequences and cut DNA approximately 1-20 bp outside the restriction site
• separate domains for DNA sequence recognition and DNA cleavage

Type III

• Smallest R-M system
• Contain 2 subunits:
• mod → modification methylase alone
• res → no enzymatic activity without mod
• Restriction requires ATP i.e. ATP is not hydrolyzed → reaction stimulated by Ado-Met (SAM)
• Restriction sites are asymmetrical and have a single N6-Methylated adenosyl on 1 strand and nothing on the other when modified
• 4 members:
• EcoP1 and EcoP15 (from P1 prophage)
• complementary
• 2 structural genes
• res → highly conserved over its entire length and is required only for restriction
• mod → mosaic of conserved and non-conserved sequences and is required for recognition of DNA for its restriction and modification as well as for catalyzing the modification
• HinfIII (H. influenzae)
• StyLTI (Salmonella)
• Restriction requires 2 restriction sites that are in inverse direction
• sites that are in direct orientation (for example, all unmodified sites of freshly replicated DNA) can only be modified but are not subject to restriction

Type IV

• Restriction system specific for modified DNA
• Examples:
• DpnI
• from S. pneumoniae
• requires a methylated substrate
• cleaves GATC only if A is methylated
• cannot act against a single-stranded DNA
• cannot cleave a hemi-methylated recognition site
• DpnII
• recognizes and cleaves GATC only if it is not methylated
• cannot act against a single-stranded DNA
• cannot cleave a hemi-methylated recognition site
• In E. coli:
• the enzymes recognize and cut DNA carrying the DNA modification signature of other strains and are therefore not accompanied by the equivalent of a classical modification enzyme
• 3 Mcr systems prevalent (Mcr = methyl-cytosine resisting)
• McrA
• McrBC
• Mrr