How does RNase H recognize a DNA.RNA hybrid?

Nakamura, Haruki; Oda, Yasushi; Iwai, Shigenori; Inoue, Hideo; Ohtsuka, Eiko; Kanaya, Shigenori; Kimura, Shigenobu; Katsuda, Chie; Katayanagi, Katsuo; Morikawa, Kosuke

doi:10.1073/pnas.88.24.11535

Cited by 206 publications

(148 citation statements)

References 26 publications

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“…Numerous conflicting proposals have been made regarding the number of metal ions necessary for activity and the precise catalytic mechanism of ribonuclease H (5,6,13,15,25,28,37), but a consensus is developing that a single metal is required for activity. It is unclear which co-crystal (Mg 2ϩ or Mn 2ϩ ) most closely approximates the position of the metal in Site 1 during catalysis on a nucleic acid.…”

Section: Discussionmentioning

confidence: 99%

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

Goedken

Marqusee

2001

Journal of Biological Chemistry

123

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ions are seen to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70 make direct (inner sphere) coordination contacts to the first (activating) metal, whereas residues Asp-10 and Asp-134 make direct contacts to the second (attenuating) metal. This structure is consistent with biochemical evidence suggesting that two metal ions may bind RNase H but liganding a second ion inhibits RNase H activity.The RNase H class of enzymes cleaves the RNA moiety of RNA⅐DNA hybrids in a divalent cation-dependent manner leaving 5Ј-phosphate and 3Ј-hydroxyl products. RNase H proteins are found in a wide variety of organisms ranging from bacteria to vertebrates (for review see Refs. 1 and 2). From a medical perspective, the most significant function of RNase H is its critical action in the life cycle of retroviruses such as the human immunodeficiency virus (HIV).1 This activity arises from the C-terminal region of reverse transcriptase. The RNase H domain from HIV is homologous to other members of this class including RNase HI from Escherichia coli. Because inhibiting RNase H activity prohibits production of infectious virions (3), understanding the function and mechanism of RNase H is an important avenue toward the development of anti-retroviral compounds.RNase H requires divalent cations (either Mg 2ϩ or Mn 2ϩ ) for catalysis. Five conserved residues form the metal-binding active site of RNase H (Fig. 1a). Two RNases H with metal ions bound have been studied by x-ray crystallography. E. coli RNase HI binds a single Mg 2ϩ ion via three carboxylates (Asp-10, Glu-48, and Asp-70) that form Site 1 (4, 5), whereas the HIV RNase H domain shows two Mn 2ϩ ions, one in a position similar to Site 1 and another (Site 2) liganded by the equivalents of Asp-10 and Asp-134 (6). Mutations in RNases H, which eliminate Mg 2ϩ -dependent activity, often allow retention of Mn 2ϩ -dependent activity (7-11). This, together with the different stoichiometry observed via crystallography, raises the possibility of alternate binding modes and/or catalytic requirements for these metals. In E. coli RNase HI, conservative mutation of the residues comprising Site 1 (Asp-10, Glu-48, and Asp-70) eliminates activity (12), whereas mutations of the conserved histidine (His-124) (13) and aspartate (Asp-134) of Site 2 (14) have smaller effects on catalysis. Despite structural efforts to identify additional weaker binding sites (5), no metal binding to Site 2 in E. coli RNase HI has been reported.Our laboratory has recently proposed a model for the metal dependence of E. coli RNase HI termed an "activation/attenuation mechanism" (15). In this model, a single metal bound at Site 1 is required for catalysis, and the binding of a second metal at Site 2 reduces catalysis ϳ100-fold. In this report, we present a high resolution crystal structure of E. coli RNase HI in complex with Mn 2ϩ ions. These data demonstrate that the active site of E. coli RNase HI can bind two Mn 2ϩ ions in the sites predicted by the activation/attenuation hypothesis. Furthermore, we s...

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Section: Discussionmentioning

confidence: 99%

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

Goedken

Marqusee

2001

Journal of Biological Chemistry

123

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“…The precise interactions between RNase H and the heteroduplex and the conformation of the DNA/RNA duplex at the active site of the enzyme have been the subject of numerous investigations over the last 15 years (see for example refs 31-34 and cited literature). The enzyme was expected to use the availability of 2′-hydroxyl groups in the RNA strand and the absence thereof in the DNA strand to discriminate between DNA/RNA and RNA/RNA duplexes (31). On the basis of NMR investigations in solution, it was concluded that sugars of the DNA strand in the hybrid duplex adopted Eastern pucker.…”

mentioning

confidence: 99%

2‘-Fluoroarabino- and Arabinonucleic Acid Show Different Conformations, Resulting in Deviating RNA Affinities and Processing of Their Heteroduplexes with RNA by RNase H^,

Sarkhel

Wilds

et al. 2006

Biochemistry

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2′-Deoxy-2′-fluoro-arabinonucleic acid (FANA) and arabinonucleic acid (ANA) paired to RNA are substrates of RNase H. The conformation of the natural DNA/RNA hybrid substrates appears to be neither A-form nor B-form. Consistent with this, the conformations of FANA and ANA were found to be intermediate between the A-and B-forms. However, FANA opposite RNA is preferred by RNase H over ANA, and the RNA affinity of FANA considerably exceeds that of ANA. By investigating the conformational boundaries of FANA and ANA residues in crystal structures of A-and B-form DNA duplexes at atomic resolution, we demonstrate that FANA and ANA display subtle conformational differences. The structural data provide insight into the structural requirements at the catalytic site of RNase H. They also allow conclusions with regard to the relative importance of stereoelectronic effects and hydration as modulators of RNA affinity.

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“…This glutamine is conserved in the RNase H domain of HIV-1 reverse transcriptase [14], and its counterpart of Thermus thermophilus RNase H is histidine [40], which also can act as a proton donor in a hydrogen bond. The mutation of Gln-72 to alanine, which resulted in a reduced affinity for the substrate [24], also suggests its contribution to the substrate recognition. At position + 1, the backbone amides of Cys-13 reside close to the 2'-hydroxyl.…”

Section: Construction Of a Tertiary Structure Model Of The Enzyme-mentioning

confidence: 99%

“…DNA hybrid duplex was constructed in a manner similar to those described previously [11,24]. The hybrid duplex structure [32] and the crystal structure of RNase HI in complex with a Mg 2÷ ion [12] were manipulated for docking on a computer graphics screen (HYDRA, Polygen).…”

Section: Model Buildingmentioning

confidence: 99%

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Recognition of 2′‐hydroxyl groups by Escherichia coli ribonuclease HI

et al. 1995

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In order to investigate the hydrogen-bonding interactions between Escherichia coli ribonuclease HI and the 2'-hydroxyl functions of the substrate, oligonucleotide duplexes containing 2'-amino-2'-deoxyuridine or 2'-fluoro-2'-deoxyuridine at a specific site were used, and their affinities for the enzyme were determined by kinetic analyses. The results indicate that the hydroxyl groups of the nueleoside 3'-adjacent to the cleaved phosphodiester linkage and the second nucleoside 5' to the cleaved phosphodiester act as both a proton donor and an acceptor and as a proton acceptor, respectively, in the enzyme-substrate complex. A molecular model was constructed using the interactions derived from the results.

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How does RNase H recognize a DNA.RNA hybrid?

Cited by 206 publications

References 26 publications

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

2‘-Fluoroarabino- and Arabinonucleic Acid Show Different Conformations, Resulting in Deviating RNA Affinities and Processing of Their Heteroduplexes with RNA by RNase H^,

Recognition of 2′‐hydroxyl groups by Escherichia coli ribonuclease HI

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How does RNase H recognize a DNA.RNA hybrid?

Cited by 206 publications

References 26 publications

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

2‘-Fluoroarabino- and Arabinonucleic Acid Show Different Conformations, Resulting in Deviating RNA Affinities and Processing of Their Heteroduplexes with RNA by RNase H,

Recognition of 2′‐hydroxyl groups by Escherichia coli ribonuclease HI

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2‘-Fluoroarabino- and Arabinonucleic Acid Show Different Conformations, Resulting in Deviating RNA Affinities and Processing of Their Heteroduplexes with RNA by RNase H^,