This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tímár, E.
Right arrow Articles by Kiss, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tímár, E.
Right arrow Articles by Kiss, A.

 Previous Article  |  Next Article 

Journal of Bacteriology, December 2008, p. 8003-8008, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00754-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants{triangledown}

Edit Tímár, Pál Venetianer, and Antal Kiss*

Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary

Received 27 May 2008/ Accepted 29 September 2008

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.

* Corresponding author. Mailing address: Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, 6726 Szeged, Temesvári krt. 62, Hungary. Phone: (36) 62 599 630. Fax: (36) 62 433 506. E-mail: kissa{at}brc.hu

{triangledown} Published ahead of print on 10 October 2008.

Journal of Bacteriology, December 2008, p. 8003-8008, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.00754-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»