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What are the effects of the bacterial DNA restriction-modification systems on cloning
and manipulations of DNA in E. coli?
Restriction-Modification (R-M) systems in E. coli evolved to protect bacteria
from invading foreign DNA such as bacteriophage genomes. These R-M systems can be split
into two broad classes.
- Those that protect bacterial DNA from restriction (degradation) by modification
(methylation) of specific sequences that are recognized by the corresponding restriction
enzymes of the R-M system.
- Those that only cut DNA bearing foreign modifications. The host DNA is then protected
from cleavage by the restriction enzymes of this system by virtue of it not being
modified.
The first type include the classic R-M systems as exemplified by the type I and type II
restriction and modification systems. Type II include the typical restriction enzymes used
in molecular biology. However, for cloning of foreign DNA sequences in E. coli,
the most important is the type I system encoded by the hsdR, hsdM and hsdS
genes. The products of all three genes form a multimeric enzyme capable of cleaving or
methylating a particular target sequence (5´-AAC[N6]GTGC-3´ in E. coli
K12 strains) (1,2). If the target sequence is hemimethylated (only one strand is
methylated as occurs during DNA replication of the host chromosome), then the enzyme acts
as a methylase thus protecting the sequence from cleavage. If the sequence is not
methylated on any strand, then the enzyme acts as a restriction endonuclease and cuts the
target sequence. Typically DNA from another organism will not be methylated at this target
sequence and will be degraded when introduced into a strain that is wildtype for all three
genes. As the hsdR gene is required only for endonuclease cleavage of the target
sequence (hsdM and hsdS are necessary and sufficient for methylation of
the target sequence), E. coli strains that are mutated for hsdR have the
so-called restriction minus, modification plus phenotype (r–, m+).
This means they can be used to clone foreign DNA that is not methylated at the target
sequence. In fact, when such DNA is propagated in this genetic background, the target
sequence becomes methylated, which then allows it to be grown in a strain that is wildtype
for all three hsd genes. Examples of E. coli strains available from Promega with
this phenotype (r–, m+) include JM109
(Glycerol Stock: Cat.#
P9751; Competent Cells: Cat.#
L1001 and L2001); glycerol stocks of: JM109(DE3)(*) (Cat.#
P9801), KW251 (Cat.#
T3571), LE392 (Cat.#
K9981), NM538 (Cat.#
D1571) and NM539 (Cat.#
D1581). Mutations in either the hsdM or hsdS genes result in a
restriction-modification minus phenotype (r–, m–). DNA
propagated in this genetic background is not subject to endonuclease cleavage but
similarly is not methylated. Consequently, plasmid DNA isolated from such a strain cannot
be introduced into a strain that is wildtype for hsdR, hsdM and hsdS
(r+, m+) as it will be degraded. It first has to be passed through a
restriction minus modification plus strain such as JM109 to protect it from cleavage. An
example of where this can be an issue is the GeneEditor™
in vitro Site-Directed Mutagenesis System(*)
(Cat.# Q9280). The
mismatch repair-deficient strain used in this system (BMH 71-18 mutS) is wildtype
for hsdR, hsdM and hsdS genes and therefore will degrade any
plasmid DNA that has been grown in a methylation minus strain of E. coli such as HB101.
Plasmids grown in strains that are wildtype for methylation (such as JM109 and DH5a™) are protected from such degradation. Thus, before
transforming a plasmid into a different strain of E. coli, it is important to
consider the hsdR, hsdM and hsdS genotype of the strain that
the plasmid was grown in as well as the genotype of the strain into which you plan to
transform it.
The Dam (DNA adenine methyltransferase) and Dcm (DNA cytosine methyltransferase)
modification systems methylate adenines and cytosines located within specific recognition
sequences (5´-GATC-3´ for Dam and the second cytosine of 5´-CCA/TGG-3´
for Dcm) (3). The biological role of these modifications does not appear to be to protect
bacterial chromosomal DNA from a corresponding restriction endonuclease within the
bacterium (such as with the class I R-M systems). However, the effect of these
modifications is to prevent certain restriction enzymes used in molecular biology from
cutting their corresponding target sequence in plasmid DNAs. This can happen if the
restriction enzyme recognition sequence encompasses the methylation sequence or overlaps
the methylation sequence (the methylated adenine or cytosine usually residing within the
restriction enzyme recognition sequence). For example, the restriction enzyme Xba
I will not cut its recognition sequence (5´-TCTAGA-3´) when the last adenine is
methylated. For obvious reasons this can be a problem if one is not able to cut a
particular site. This can be overcome by transforming the plasmid into a strain that is
mutated for dam, or dcm or both genes.
The second type of restriction-modification system consists of those that direct
endonuclease cleavage to DNA targets that are methylated on certain sequences (2). In E.
coli there are two methyl-cytosine restricting systems called McrA and McrBC. In
addition, there is also a methylated adenine recognition and restriction system called
Mrr. Neither of these systems cleaves DNA that has been methylated by the type I (encoded
by the hsdRMS genes), Dcm or Dam systems. Both the McrA and McrBC systems
restrict hemimethylated DNA as well as DNA methylated on both strands. DNA that has been
methylated by the Sss I methylase (methylates the cytosine of the CG
dinucleotide) and the Hpa II methylase (methylates the second cytosine of the
sequence 5´-CCGG-3´) are restricted by by the McrA system (4,5). The McrBC system
cleaves DNA methylated at the cytosines of the sequence (G/A)C (6).
As mammalian DNA tends to be methylated at cytosines in CG sequences and plant DNA at
cytosines in CNG sequences, it is recommended to use strains that are mutated for these
systems to increase the recovery of genomic DNA propagated in E. coli during
cloning experiments. The Mrr system cleaves DNA methylated at adenines and has also been
reported to restrict DNA methylated at cytosines. However, there is no apparent consensus
sequence for recognizing these methylated bases by this system. As with the McrA and McrB
systems, strains that are mutated in the Mrr system should be considered when cloning DNA
from other organisms as plants and mammals usually contain some degree of methylation that
can be recognized by these systems.
References
- Yuan, R. and Hamilton, D.L. (1984) In: DNA Methylation: Biochemistry and Biological
Significance, Razin, A., Cedar, H., and Riggs, A.D., eds., Springer-Verlag, New York.
- Redaschi, N. and Bickle, T.A. (1996) In: Escherichia coli and Salmonella:
Cellular and Molecular Biology, Second Edition, Neidhardt, F.C., ed., ASM Press,
Washington, D.C.
- Marinus, M.G. (1996) In: Escherichia coli and Salmonella: Cellular and
Molecular Biology, Second Edition, Neidhardt, F.C., ed., ASM Press, Washington, D.C.
- Raleigh, E.A. and Wilson, G. (1986) Escherichia coli K-12 restricts DNA
containing 5-methylcytosine. Proc. Natl. Acad. Sci. USA 83,
9070-9074.
- Kelleher, J.C. and Raleigh, E.A. (1991) A novel activity in Escherichia coli K-12
that directs restriction of DNA modified at CG dinucleotides. J. Bacteriol. 173,
5220-5223.
- Raleigh, E.A. (1992) Organization and function of the mcrBC genes of Escherichia
coli K-12. Mol. Microbiol. 6, 1079-1086.
GeneEditor is a trademark of Promega Corporation.
DH5a is a trademark of Life Technologies, Inc.
*Products may be covered by pending or issued patents. Please visit
our patent and trademark web page for more information.
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