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Abstract
2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyri- midine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5, 6-tetrahydropyrimidine (THP(B) or ectoine) are now recognized as ubiquitous bacterial osmoprotectants. To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases. THP(A) completely arrested the cleavage of plasmid and bacteriophage lambda DNA by EcoRI endonuclease at 0.4 mM and the oligonucleotide (d(CGCGAATTCGCG))2 at about 4.0 mM. THP(B) was 10-fold less effective than THP(A), whereas for betaine and proline, a notable inhibition was observed only at 100 mM. Similar effects of THP(A) were observed for all tested restriction endonucleases, except for SmaI and PvuII, which were inhibited only partially at 50 mM THP(A). No effect of THP(A) on the activity of DNase I, RNase A, and Taq DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibited the EcoRI-(d(CGCGAATTCGCG))2 complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carboxy group significantly decreased the activity of THPs, suggesting that their zwitterionic character is essential for the inhibition effect. Possible mechanisms of inhibition, the role of THPs in the modulation of the protein-DNA interaction, and the in vivo relevance of the observed phenomena are discussed.
Highlights
2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are recognized as ubiquitous bacterial osmoprotectants
To evaluate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA interaction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleases
To assess whether polyamines could be remedial for the restriction system inhibited by THPs, we studied the effect of spermine on EcoRI binding to and cleavage of ((dCGCGAATTCGCG))2 oligonucleotide, arrested by THP(A)
Summary
2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydroxyectoine) and 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine (THP(B) or ectoine) are recognized as ubiquitous bacterial osmoprotectants. The role and activities of THPs are of special interest as they represent a limited group of osmoprotectants that are synthesized de novo, in the bacterial cell, in contrast to those transported from the medium [6] Their synthesis in a number of Streptomyces strains as a response to increased salinity and elevated temperature was recently described [7]. THP molecules share similarity in structure and geometry with pyrimidine bases and have the zwitterionic character, which was recently shown to be responsible for modification of DNA electrostatic interaction with counterions by a number of osmolytes [25] These notions led us to suggest that the impact of osmoprotectants, and THPs in particular, on protein-DNA interaction is, likely, underestimated. The impact of osmolytes on the sequence-specific interaction of proteins with DNA (e.g. restriction endonucleases and transcription factors) could be even more complex and remains to be elucidated
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