KINETIC AND STOICHIOMETRIC ANALYSIS FOR THE BINDING OF ESCHERICHIA-COLI RIBONUCLEASE HI TO RNA-DNA HYBRIDS USING SURFACE-PLASMON RESONANCES

To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase H I to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) u sing surface plasmon resonance (BIAcore((TM))). The k(on) and k(off) v alues for the binding of the enzyme to the 36 bp substrate were 1.5 x 10(6) M-1 s(-1) and 3.2 x 10(-2) s(-1), respectively, Similar values w ere obtained with the shorter substrates, Using uncleavable 2'-O-methy lated RNA-DNA substrates, values for h(on) and h(off) were 2.1 x 10(5) M-1 s(-1) and 1.3 x 10(-1) s(-1) in the absence of Mg2+ that were fur ther reduced in the presence of Mg2+ to 7.4 x 10(3) M-1 s(-1) and 2.6 x 10(-2) s(-1). Kinetic parameters similar to the wild-type enzyme wer e obtained using an active-site mutant enzyme, Asp(134) replaced by Al a, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby d istinguishing between poor catalysis and inability to bind to the subs trate. Stoichiometric analyses of RNase HI binding to substrates of 18 , 24, 30, and 36 bp are consistent with previous reports suggesting th at RNase HI binds to 9-10 bp of RNA DNA hybrid.