STUDIES OF THE INTERACTIONS BETWEEN ESCHERICHIA-COLI RIBONUCLEASE HI AND ITS SUBSTRATE

Ribonuclease H (RNase H) recognizes a DNA-RNA hybrid duplex and cataly zes the hydrolysis of the phosphodiester linkages in only the RNA stra nd, Previously we developed a method to cleave RNA in a sequence-depen dent manner using RNase H and a complementary oligonucleotide containi ng 2'-O-methylribonucleosides. Since cleavage is restricted to a singl e site by the modified complementary strand, this system allows kineti c analysis of the RNase H reaction. We describe an investigation of th e interactions between RNase HI from Escherichia coli and its substrat e, and between the: substrate and a metal ion using synthetic oligonuc leotide duplexes modified at the cleavage site in combination with the 2'-O-methylribonucleotides. Firstly, the base moiety was changed to i nter fere with enzyme binding in either the major or minor groove. Whe n 2-N-methylguanine was incorporated into the cleavage site, the K-m v alue for this substrate, containing a methyl group in the minor groove , was 20-fold larger than that for the unmodified substrate, whereas 5 -phenyluracil, with a phenyl group residing in the major groove of the duplex, did not affect the affinity. Secondly, the phosphodiester lin kage at the cleavage site was changed into a phosphorothioate with a d efined configuration. Only the R(P) isomer was cleaved at this site in the presence of Mg2+ or Cd2+ These results suggest that the enzyme, b ut not the metal ion, interacts with the phosphate residue at the clea vage site. Thirdly, the 2'-position of the nucleoside on the 5'-side o f the scissile phosphodiester was modified. Alteration of the 2'-hydro xyl function into an amino, fluoro or methoxy group, or removal of thi s 2'-hydroxyl group, did not affect the affinity for the enzyme, but r educed the reaction rate. An outer sphere interaction of a metal ion w ith the 2'-hydroxyl group is suggested.