In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants

Edit Tímár, Pál Venetianer, A. Kiss

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The SinI DNA methyltransferase, a component of the SinI restriction-modification system, recognizes the sequence GG(A/T)CC and methylates the inner cytosine to produce 5-methylcytosine. Previously isolated relaxed-specificity mutants of the enzyme also methylate, at a lower rate, GG(G/C)CC sites. In this work we tested the capacity of the mutant enzymes to function in vivo as the counterpart of a restriction endonuclease, which can cleave either site. The viability of Escherichia coli cells carrying recombinant plasmids with the mutant methyltransferase genes and expressing the GGNCC-specific Sau96I restriction endonuclease from a compatible plasmid was investigated. The sau96IR gene on the latter plasmid was transcribed from the araBAD promoter, allowing tightly controlled expression of the endonuclease. In the presence of low concentrations of the inducer arabinose, cells synthesizing the N172S or the V173L mutant enzyme displayed increased plating efficiency relative to cells producing the wild-type methyltransferase, indicating enhanced protection of the cell DNA against the Sau96I endonuclease. Nevertheless, this protection was not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the N172S and V173L mutants. Elevated DNA ligase activity was shown to further increase viability of cells producing the V173L variant and Sau96I endonuclease.

Original languageEnglish
Pages (from-to)8003-8008
Number of pages6
JournalJournal of Bacteriology
Volume190
Issue number24
DOIs
Publication statusPublished - Dec 2008

Fingerprint

Methyltransferases
Plasmids
DNA
DNA Restriction Enzymes
Enzymes
DNA Restriction-Modification Enzymes
5-Methylcytosine
DNA Ligases
Arabinose
Cytoprotection
Endonucleases
Cytosine
Helper-Inducer T-Lymphocytes
Methylation
Genes
Cell Survival
Escherichia coli
GGNCC-specific type II deoxyribonucleases

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants. / Tímár, Edit; Venetianer, Pál; Kiss, A.

In: Journal of Bacteriology, Vol. 190, No. 24, 12.2008, p. 8003-8008.

Research output: Contribution to journalArticle

Tímár, Edit ; Venetianer, Pál ; Kiss, A. / In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants. In: Journal of Bacteriology. 2008 ; Vol. 190, No. 24. pp. 8003-8008.
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