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When is making unmethylated DNA necessary?
For one of three reasons:
- To cut plasmid DNA (grown in E. coli) with a restriction endonuclease sensitive to E.
coli K-12 methylation patterns (Dam [G meATC] or Dcm [C meC(A/T)GG]). Examples are BclI,
TGATCA or ScrFI (CCNGG). For a list of endonucleases
sensitive to these, please click here.
- To transform shuttle plasmid DNA grown in E. coli into a strain
of eubacteria or archaea that restricts DNA with this methylation. Genera reported to restrict methylated
DNA include Streptomyces, Acholeplasma, Bacillus, Brevibacterium, Corynebacterium and Haloferax (1-6).
- To study the effects of methylation itself, e.g. on expression or DNA repair (7-12).
What strains can be used for this?
Any otherwise suitable strain that lists dam and dcm in its genotype.
- For routine use, NEB offers ready to use competent cells, dam-/dcm- competent E.
coli (NEB# C2925H/I). We also supply the strain
ER2925 (#E4109S), free with an order or for the cost
of shipping.
- For M13 or when LacI repression is needed , JM110 is available from ATCC (ATCC
Web site). JM110 carries an F' that bears lacIq DEL(lacZ)M15, and is thus able to grow
M13 derivatives and, repress high-copy lac promoters, and
complement a-fragment expressed by plasmids and phagemids derived
from pUC vectors.
- When recombination between repeated sequences is a problem , GM2929 is an alternative (available
from the E. coli Genetic Stock Center at Yale University; (CGSC
Web site). This strain is ER2925 recF. Strains with recF are as defective in plasmid-by-plasmid
recombination as strains with recA (9). Mutations in recA can't be combined with dam because
of DNA repair problems (13, 14).
- A review of available Dam- and Dcm- strains can be found in ref. 15.
- Note that all enteric bacteria express Dam. (So do cyanobacteria and Haemophilus spp refs.
16, 17). Derivatives of E. coli B, such as BL21(DE3), T7 Express (NEB#
C2566H/I) and NEB Express (NEB# C2523H/I), naturally
lack Dcm. Most strains used by molecular biologists are derivatives of K-12, and possess both Dam and
Dcm unless otherwise noted.
Are there any special considerations when working with dam-/dcm- strains?
- We do not recommend these strains as a host for primary cloning/ligation. The dam mutation
can result in an increased mutation rate in the cell (18, 19). This is because Dam affects mismatch repair
and the timing of the initiation of DNA replication. The dam mutation
will also reduce the transformation efficiency of competent cells. DNA should be maintained in a Dam+
strain unless there is a specific need for DNA free of Dam or Dcm methylation.
- C2925 has a low but noticeable frequency of kanamycin resistant
mutations. We have observed a low, variable number of spontaneous Kan resistance mutants in this strain,
and also in another commercially available dam- dcm- strains.
In both strains, tiny colonies appear on Kan plates at a frequency ranging from 1 in 106 to
1 in 107 cells. These tiny colonies will not grow upon restreaking on a fresh Kan plate. This
level of background resistance should not be a problem for routine transformation of plasmids carrying
KanR. The reason for the strain-dependent appearance of incomplete KanR is not
known. Mutations in tufG can yield resistance to low levels of
kanamycin (ref. 20). There are also known interactions between ribosomal mutations leading to streptomycin
resistance (rpsL, mutated in both tested strains) and tufG.
Hypothetically, a defect in drug transport could reduce the effective intracellular kanamycin concentration,
enabling low-level resistance mechanisms to be effective. Both tested strains carry a tsx mutation;
this gene encodes a channel protein in E. coli that functions
in permeation of nucleosides across the outer membrane. However, this porin is not known to play a role
in aminoglycoside permeation.
Are there other tips for making unmethylated DNA?
- Good microbiological practice. Always begin with a single colony when growing the strain to make
competent cells. Dam+ revertants can arise (expected revertant frequency in an overnight culture < ~106)
and when they do they will have a selective advantage and will accumulate during serial mass transfer.
One subculture to get the cells to exponential phase is acceptable.
- Maintaining the dam mutation: The dam mutation in C2925 is the result of insertion
by the CamR transposon Tn9. Although not essential, it may be beneficial to maintain selection
for the insertion by keeping the strain on chloramphenicol (15 µg/ml). This does not mean that
serial transfers are OK if you maintain selection, however.
- Spectinomycin amplification of plasmid copy number: Some plasmids (colE1, pBR322, pACYC184 and
177, but NOT pUC derivatives) can be obtained in higher yields from C2925, as from any other strain,
by using an
"amplification" procedure involving inhibition of protein synthesis (15). Since C2925 is CamR,
protein synthesis is not inhibited by the chloramphenicol called for in the original procedure, so use
spectinomycin instead. DO NOT TRY TO GROW C2925 ON SPECTINOMYCIN. The procedure cited is based on the
repression of plasmid number by the plasmid Rop protein or analogue, and on the independence of plasmid
but not host replication initiation from new protein synthesis. Basically, the host chromosome stops
replicating when protein synthesis is inhibited, but the plasmid keeps replicating and will continue
to replicate to very high copy if the Rop protein is not present. pUC and other very high copy number
plasmids are already missing Rop and will not give improved yields with this procedure.
References:
- Holmes, M. L., Nuttall, S. D., and Dyall-Smith, M. L. (1991): Construction and use of halobacterial shuttle
vectors and further studies on Haloferax DNA gyrase. J. Bacteriol. 173, 3807-3813.
- Macaluso, A., and Mettus, A.-M. (1991): Efficient transformation of Bacillus thuringiensis requires
nonmethylated plasmid DNA. J. Bacteriol. 173, 1353-1356.
- MacNeil, D. J. (1988): Characterization of a unique methyl-specific restriction system in Streptomyces
avermitilis. J. Bacteriol. 170, 5607-5612.
- Sladek, R. L., Nowak, J. A., and Maniloff, J. (1986): Mycoplasma restriction: identification of a new
type of restriction specificity for DNA containing 5-methylcytosine. J. Bacteriol. 165, 219-225.
- Vertes, A. A., Inui, M., Kobayashi, M., Kurusu, Y., and Yukawa, H. (1993): Presence of mrr- and mcr-
like restriction systems in coryneform bacteria. Res. Microbiol. 144, 181-185.
- Chmuzh, E.V., Kashirina, J.G., Tomilova, J.E., Mezentseva, N.V., Dedkov, V.S., Gonchar, D.A., Abdurashitov,
M.A., Degtyarev, S.K. (2005):A Novel Restriction endonuclease BisI from Bacillus subtilis T30,
recognizes a methylated DNA sequence 5'- G(m5C)^NGC-3'. Biotekhnologiya 3: 22-26.
- Marinus, M. G. (1987): DNA methylation in Escherichia coli. Ann. Rev. Genet. 21, 113-131.
- Noyer-Weidner, M., and Trautner, T. A. (1993): Methylation of DNA in prokaryotes. Exs 64, 39-108.
- Lahue, R. S., Au, K. F., and Modrich, P. (1989): DNA mismatch correction in a defined system. Science 245,
160-164.
- Kolodner, R. (1985): J. Bacteriol. 163, 1060.
- Marinus, M.G. (2005): Dr. Jekyll and Mr. Hyde: How the MutSLH Repair System Kills the Cell, pp. 413-429.
In Higgins, N.P (ed.) The Bacterial Chromosome. ASM Press, Washington DC.
- Robertson KD, Jones PA. (1997): Dynamic interrelationships between DNA replication, methylation, and
repair. Am J Hum Genet. 61:1220-4.
- Marinus, M. G., and Morris, B. (1975): Biological function for 6-methyladenine residues in the DNA of
Escherichia coli K12. J. Mol. Biol. 85, 309-322.
- McGraw, B., and Marinus, M. G. (1980): Isolation and characterization of Dam+ revertants and suppressor
mutations that modify secondary phenotypes of dam-3 strains of Escherichia coli K-12. Mol. Gen. Genet. 178,
309-315.
- Palmer, B. R., and Marinus, M. G. (1994): The dam and dcm strains of Escherichia coli--a review. Gene 143,
1-12.
- Brooks, J. E., Blumenthal, R. M., and Gingeras, T. R. (1983): The isolation and characterization of the
Escherichia coli DNA adenine methylase (dam ) gene. Nucleic Acids Res. 11, 837-851.
- Barbeyron, T., Kean, K., and Forterre, P. (1984): DNA adenine methylation of GATC sequences appeared
recently in the Escherichia coli lineage. J. Bacteriol. 160, 586-590.
- Glickman, B. W, and Radman, M. (1980) Escherichia coli mutator mutants deficient in methylation-instructed
DNA mismatch correction. Proc. Nat. Acad. Sci USA 77, 1063-1067.
- Marinus, M. G., Carraway, M., Frey, A. Z., Brown, L., and Arraj, A. J. (1983) Insertion mutants in the
dam gene of Escherichia coli K-12 . Mol. Gen. Genet. 192, 288-289.
- Kurland, C. G., Hughes, D. and Ehrenberg, M. Limitations of Translational Accuracy (1996) in Escherichia
coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington, DC
- Sambrook, J. and Russell, D. (2001) Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory.
Cold Spring Harbor, N.Y., Volume 1, pp. 1.38-1.41.
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