Affiliation of authors: Institut Nationale de la Santé et de la Recherche Médicale (INSERM) U318, CHU Michallon, Grenoble, France.
Correspondence to: Didier Wion, Ph.D., INSERM U318, CHU Michallon, 38043 Grenoble France (e-mail: didier.wion{at}ujf-grenoble.fr).
In Escherichia coli, a base substitution hotspot occurs in
the second C of CC(A/T)GG sequences (where C = cytosine, A = adenine, T
= thymine, and G = guanine) (1). It is the consequence of
the
E. coli dcm (DNA cytosine methylation) methylase, which
modifies this cytosine to 5-methylcytosine (2). Then,
spontaneous deamination of 5-methylcytosine to thymine introduces a G:C
A:T transition. Because of its bacterial origin, this mutational
hotspot will also be found in any plasmid DNA used in gene therapy.
Hence, plasmid preparations may contain a small percentage of mutated
copies that will not be detected by DNA sequencing if the transition
occurred late in the amplification step of plasmid. These mutated
plasmids, even if they are quantitatively negligible, could have
dramatic biologic consequences if they generate recombinant proteins
with a dominant gain of aberrant functions (3). For instance,
we have found seven CC(A/T)GG motifs in the p53-coding sequence. The
corresponding G:C
A:T transitions will give rise to either
proline
leucine substitutions (codons 58, 278, 301, and 359) or
nonsense mutations (codons 104 and 354). It is interesting that proline
leucine substitutions at codons 278 and 301 have already been
detected in human tumors (4-6). However, hotspots in E.
coli will not necessarily overlap hotspots in eukaryotes. It is
noteworthy that, in humans, 5-methylcytosine is mainly found at the
dinucleotide CG and not in the CC(A/T)GG sequence. Thus, the absence of
G:C
A:T transitions at codons 58, 104, 354, and 359 in human
tumors does not necessarily mean that they are biologically neutral but
may suggest that these codons are not hotspots in humans. The possible
biologic consequences of these mutations are, therefore, presently
unknown. For all of these reasons, we suggest that the potential hazard
of methylated CC(A/T)GG motifs should be evaluated for every plasmid
encoding a therapeutic protein for which dominant mutant forms have
been described.
REFERENCES
1 Coulondre C, Miller JH, Farabaugh PJ, Gilbert W. Molecular basis of base substitution hotspots in Escherichia coli. Nature1978 ;274:775-80.[Medline]
2 Palmer BR, Marinus MG. The dam and dcm strains of Escherichia colia review. Gene 1994;143:1-12.[Medline]
3 Berger F, Canova C, Benabid AL, Wion D. Are sequences of plasmid DNA used in gene therapy erroneous? Nat Biotechnol 1999;17:517.
4 Yin J, Harpaz N, Tong Y, Huang Y, Laurin J, Greenwald BD, et al. p53 point mutations in dysplastic and cancerous ulcerative colitis lesions. Gastroenterology 1993;104:1633-9.[Medline]
5 Kusser WC, Miao X, Glickman BW, Friedland JM, Rothman N, Hemstreet GP, et al. p53 mutations in human bladder cancer. Environ Mol Mutagen1994 ;24:156-60.[Medline]
6 Oda H, Nakatsuru Y, Ishikawa T. Mutations of the p53 gene and p53 protein overexpression are associated with sarcomatoid transformation in renal cell carcinomas. Cancer Res 1995;55:658-62.[Medline]
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