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E.coli Host Restriction and Modification Systems

System

Description

Effect on cloning experiments
EcoKI,  EcoBI Type I restriction- modification systems are encoded by host hsdR, hsdM and hsdS genes (1). Products of all three genes form a multisubunit enzyme possessing both endonucleolytic and methylation activities (1). The enzyme acts as a methylase when the recognition sequence is hemimethylated, methylating the unmodified strand. When the recognition site is completely unmodified, the enzyme acts as an enzyme and hydrolyzes DNA. In addition, products of hsdM and hsdS form a complex capable of  modifying specific DNA targets.
EcoKI recognizes the sequence 5'...AAC(N)6GTGC...3' (2), EcoBI recognizes the sequence 5'...TGA(N)8TGCT...3' (3). 		The majority of laboratory strains of E.coli are derived from wild-type strains K-12 and B, which possess the EcoKI and EcoBI restriction-modification systems, respectively. hsdS mutations abolish both DNA modification and restriction; hsdR mutants are modification-proficient but restriction-deficient. If plasmid DNA which contains an EcoKI or EcoBI recognition sequence is isolated from a strain lacking the EcoKI or EcoBI methylation function (i.e. hsdM or hsdS mutants) and is introduced into an hsdR+M+S+ recipient strain, transformation efficiency may be markedly reduced. Modification of EcoKI or EcoBI DNA targets can result in reduced cleavage by restriction enzymes whose targets overlap the recognition sites. 
Dam DNA adenine methylase is encoded by the dam gene of E.coli. It modifies the adenine residue at the N6-position on both strands within the sequence 5'...GATC (4, 5).
  • Dam methylase-modified plasmids with either the E.coli chromosomal replication origin or the pMB1 plasmid origin transform E.coli dam mutants poorly (6, 7). This effect is suppressed completely if transforming DNA is not methylated (8). Methylated or unmethylated plasmids transform dam+ strains efficiently.
  • DNA isolated from E.coli dam+ strain can be difficult to hydrolyze by restriction enzymes whose targets overlap (partially or completely) the Dam recognition site, see.
Dcm DNA cytosine methylase is encoded by the dcm gene of E.coli. It modifies the internal cytosine residue at the C5-position on both strands within the sequence 5'...CC(A/T)GG-3' (9, 10). DNA isolated from E.coli dcm+ strain can be difficult to hydrolyze by restriction enzymes whose targets overlap (partially or completely) the Dcm recognition site, see.
McrA The E.coli chromosome-encoded restriction system named McrA (for Modified Cytosine Restriction) is directed against DNA methylated at the sequence 5'...Cm5CGG, which is its only known target (11). The McrA+ phenotype of the host can interfere with the cloning of appropriately modified DNA (or genes coding for DNA modification enzymes of either CCGG or overlapping specificities). McrA phenotypes of some laboratory E.coli strains are presented in reference 12.
McrBC McrBC is the other chromosome-encoded E.coli restriction system, which, like McrA, is specific for modified cytosine. It is encoded by two neighbor genes, mcrB and mcrC (13). McrBC requires the presence of two (G/A)mC recognition elements (where mC is 5-hydroxymethylcytosine, N4-methylcytosine or 5-methylcytosine (13)) appropriately spaced in the substrate DNA (14). DNA cleavage occurs in region between two recognition elements (14). The optimal separation between elements is 55-103 base pairs (14).  The McrBC+ phenotype of the host can interfere with the cloning of appropriately modified DNA or genes coding for DNA modification enzymes of overlapping specificity. McrBC phenotypes of some laboratory E.coli strains are presented in reference 12. 
Mrr The E.coli chromosome-encoded restriction system named Mrr (for Methylated adenine recognition and restriction) acts against DNA containing both N6-methyladenine and 5-methylcytosine (15-17). No simple consensus sequence causing Mrr-associated restriction has been determined. The precise specificity of Mrr is uncertain, but it requires the presence of either modified adenine of cytosine. Thus, Mrr+ phenotype of the host can interfere with the cloning of appropriately modified DNA or genes coding for DNA modification enzymes of overlapping specificity. 

References

  1. Yuan, R., , et al. (eds.), DNA methylation, Biochemistry and Biological Significance, Springer-Verlag, New York, 11-38, 1984. 

  2. Kan, N.C., et al., J. Mol. Biol., 130, 191-209, 1979. 
  3. Lautenberger, J.A., et al., Proc. Natl. Acad. Sci. USA, 75, 2271-2275, 1978. 
  4. Hattman, S., et al., J. Mol. Biol., 126, 367-380, 1978. 
  5. Geier, G.E., Modrich, P., J. Biol. Chem., 254, 1408-1413, 1979. 
  6. Messer, W., et al., EMBO J., 4, 1327-1332, 1985. 
  7. Smith, D.W., et al., EMBO J., 4, 1319-1326, 1985.
  8. Russell, D.W., Zinder, N.D., Cell, 50, 1071-1079, 1987. 
  9. Buryanov, Y.I., et al., FEBS Lett., 88, 251-254, 1978.
  10. May, M.S., Hattman, S., J. Bacteriol., 123, 768-770, 1975. 
  11. Raleigh, E., Wilson, G., Proc. Natl. Acad. Sci. USA, 83, 9070-9074, 1986. 
  12. Raleigh, E., et al., Nucleic Acids Res., 15, 1563-1575, 1988. 
  13. Dila, D., et al., J. Bacteriol., 172, 4888-4900, 1990.
  14. Stewart, F.J., Raleigh, E.A., Biol. Chem., 379, 611-616, 1998. 
  15. Heitman, J., Model, P., J. Bacteriol., 169, 3243-3250, 1987. 
  16. Kelleher, J.E., Raleigh, E.A., J. Bacteriol., 173, 5220-5223, 1991.
  17. Waite-Rees, P.A., et al., J. Bacteriol., 173, 5207-5219, 1991.
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Updated balandžio 06, 2006 04:07