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Volume 272, Number 35, Issue of August 29, 1997 pp. 22015-22022
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

(Received for publication, October 18, 1996, and in revised form, June 25, 1997)

Mitsuru Haruki ^§ , Eriko Noguchi ^¶ , Shigenori Kanaya and Robert J. Crouch ^§

From the ^§ Laboratory of Molecular Genetics, NICHD, National Institutes of Health, Bethesda, Maryland 20892, the  Department of Material and Life Sciences, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565, and the ^¶ Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan

To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcore^TM). The k_on and k_off values for the binding of the enzyme to the 36-bp substrate were 1.5 × 10⁶ M¹ s¹ and 3.2 × 10² s¹, respectively. Similar values were obtained with the shorter substrates. Using uncleavable 2-O-methylated RNA-DNA substrates, values for k_on and k_off were 2.1 × 10⁵ M¹ s¹ and 1.3 × 10¹ s¹ in the absence of Mg²⁺ that were further reduced in the presence of Mg²⁺ to 7.4 × 10³ M¹ s¹ and 2.6 × 10² s¹. Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp¹³⁴ replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate. Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9-10 bp of RNA-DNA hybrid.

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