Effect of tetrahydropyrimidine derivatives on protein nucleic acids interaction - Type II restriction endonucleases as a model system

2-Methyl-4-carboxy,5-hydroxy-3,4,5,6-tetrahydropyrimidine (THP(A) or hydrox yectoine) and 2-methyl,4-carboxy-3, 4,5,6-tetrahydropyrimidine (THP(B) or e ctoine) are now recognized as ubiquitous bacterial osmoprotectants, To eval uate the impact of tetrahydropyrimidine derivatives (THPs) on protein-DNA i nteraction and on restriction-modification systems, we have examined their effect on the cleavage of plasmid DNA by 10 type II restriction endonucleas es. THP(A) completely arrested the cleavage of plasmid and bacteriophage A 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), where as for betaine and proline, a notable inhibition was observed only at 100 m M, Similar effects of THP(A) were observed for all tested restriction endon ucleases, 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 Tag DNA polymerase was noticed. Gel-shift assays showed that THP(A) inhibi ted the EcoRI-(d-(CGCGAATTCGCG))(2) complex formation, whereas facilitated diffusion of EcoRI along the DNA was not affected. Methylation of the carbo xy group significantly decreased the activity of THPs, suggesting that thei r zwitterionic character is essential for the inhibition effect. Possible m echanisms of inhibition, the role of THPs in the modulation of the protein- DNA interaction, and the in vivo relevance of the observed phenomena are di scussed.