PMID: 7544280Aug 1, 1995Paper

Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate

European Journal of Biochemistry
E Kanaya, Shigenori Kanaya

Abstract

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the...Continue Reading

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Citations

May 5, 2011·Journal of the American Chemical Society·Edina RostaGerhard Hummer
Jun 1, 2001·Journal of Biochemical and Biophysical Methods·E ZamaratskiJ Chattopadhyaya
May 29, 2004·Journal of Molecular Biology·Joel A ButterwickArthur G Palmer
Nov 16, 2013·PloS One·Sunghyuk LimJames B Ames
Oct 25, 2011·Journal of Molecular Biology·Sara B-M WhittakerGeoffrey R Moore
Oct 13, 2004·Journal of Structural Biology·Heather PetoR William Broadhurst
Nov 13, 2007·Biophysical Journal·Vasyl BondarenkoPei Tang
Nov 26, 2010·Journal of Molecular Biology·Carla F CervantesElizabeth A Komives
Apr 7, 2005·Journal of Molecular Biology·Mahon L MaguireR William Broadhurst
Apr 2, 2015·Scientific Reports·Stefano PersanoPier Paolo Pompa
Oct 5, 2002·Protein Engineering·Hyongi ChonShigenori Kanaya
Feb 23, 2019·Biotechnology Journal·Yujin JungHyun G Park
Jan 9, 2008·Chembiochem : a European Journal of Chemical Biology·Yan ChenWeihong Tan
Sep 25, 2002·Molecular and Cellular Probes·J RizzoP Lu

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