Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Abstract

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.

Crystal structure of RNA-DNA duplex provides insight into conformational changes induced by RNase H binding

“…20,26,27 The structure of the GAA duplex indicates that this conformation is likely induced by protein binding. RNases H are not able to cleave dsRNA, suggesting that the added flexibility of a DNA strand is required for conformational changes to the duplex upon protein binding.…”

“…By having a greater bend, the duplex can interact more favorably with RNase H due to an increased exposure of the surface area of the minor groove and preventing water molecules from diminishing hydrogen bonding interactions with the binding pockets. 26 These structural changes are induced by interactions with the protein, most notably the phosphate binding pocket of RNase H 20 , created by residues T104, N106, S147, and T148 (Fig. 3).…”

“…It has been demonstrated that the binding of B-DNA to RNase H forces the DNA to have a narrow minor groove but cleavage still does not occur. 26 Another requirement for catalysis to occur could be based on the degree of curvature of the helix. B-DNA is fairly linear with a bend less than 6 The superimposition of the 2 RNA-DNA hybrids shows the RNA strand has a relatively similar conformation, but the DNA strand has been distorted to significantly widen the major groove of the helix (see Tables 2 and 3).…”

“…Understanding how RNA-DNA hybrids are recognized and processed by RNase H enzymes is an important step in determining how enzyme dysfunction leads to disease. While structures of RNase H enzyme in complex with RNA-DNA and dsDNA duplexes have been determined, [21][22][23][24][25][26] the recognition requirements for double-stranded RNA-DNA hybrids are still unclear. Relatively few structures of RNA-DNA hybrids alone have been determined, most of which are 10 bp or less or contain polypurine or polypyrimidine tracts (PPT).…”

“…Our results show that, like many other crystallographic hybrid structures, the RNA-DNA duplex takes almost solely the A-form parameters but upon binding to RNase H, widens the major groove to widths much closer to B-DNA and decreases the curvature of the duplex, which may be key steps in the recognition and cleavage of ribonucleotides from DNA. [22][23][24]26 The RNase H protein contacts the RNA and DNA strands in the minor groove and can differentiate between the A-and B-forms of the duplex as well as the sugar geometry/moiety (ribose or deoxyribose) to ensure specificity for an RNA-DNA hybrid.…”