Metal binding and activation of the ribonuclease H domain from Moloney murine leukemia virus

Eric R. Goedken and Susan Marqusee1

Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall, Berkeley, CA 94720, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The RNase H family of enzymes degrades RNA in RNA·DNA hybrids in a divalent cation-dependent manner. RNases H from diverse sources such as Escherichia coli and human immunodeficiency virus (HIV) share homologous metal-binding active sites, and the activity of the RNase H domain of reverse transcriptase (RT) is required for retroviral replication. The isolated RNase H domain from HIV RT, however, is inactive. In contrast, the RNase H domain of Moloney murine leukemia virus (MMLV) is active, enabling functional studies. Unlike both E.coli RNase HI and HIV RT, the RNase H activity of MMLV RT shows greater activity in Mn2+ than Mg2+. We investigated the effect of mutations in five conserved active-site residues of the isolated MMLV RNase H domain. Mutations in two carboxylates eliminate metal binding while mutations in other active-site residues allow retention of metal ion affinity. Mutations that inactivate E.coli RNase HI in Mg2+ have similar effects on the Mn2+-dependent activity of MMLV RNase H. These results suggest a similar one-metal catalytic mechanism for the Mn2+- and Mg2+-dependent activities of both prokaryotic and retroviral ribonucleases H.

Keywords: divalent metals/nucleic acid hydrolysis/retrovirus/RNase H/reverse transcriptase


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ribonuclease H (RNase H) catalyzes the degradation of RNA within RNA·DNA hybrids in a divalent cation-dependent manner. The best characterized class of RNase H proteins is a large family of structurally similar enzymes found in prokaryotes, eukaryotes and in the C-terminal domains of retroviral reverse transcriptases (RTs) (Figure 1aGo). Because this activity is essential to the retroviral life cycle, RNase H offers an attractive target for anti-retroviral pharmaceuticals. The RNase H domain of RT is covalently linked to an N-terminal polymerase domain that also uses divalent metals for catalysis (Figure 1aGo). Independent expression of the RNase H domain can therefore simplify studies on its metal binding and activity. Without the polymerase domain of RT, however, the human immunodeficiency virus (HIV) RNase H domain is inactive (Becerra et al., 1990Go; Hostomsky et al., 1991Go) though it can be partially re-activated by addition of a histidine tag (Evans et al., 1991Go; Smith and Roth, 1993Go) or by large extensions into the polymerase domain (Smith et al., 1994Go). In contrast, the RNase H domain of Moloney murine leukemia virus (MMLV) RT retains activity in the absence of the polymerase domain (Schultz and Champoux, 1996Go; Zhan and Crouch, 1997Go) providing a useful retroviral model for RNase H.




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Fig. 1. Identification of putative active site residues in MMLV RNase H. (a) Amino acid sequence of Moloney murine leukemia virus reverse transcriptase aligned with E.coli RNase HI (Johnson et al., 1986Go). The MRH-175 expression construct (Goedken and Marqusee, 1998Go) begins with an exogenous initiator methionine followed by the C-terminal 175-amino acids of MMLV RT [D497–L671 of the processed MMLV RT polypeptide (Copeland et al., 1985Go) or D617–L791 of MMLV pol polyprotein (Shinnick et al., 1981Go)]. Residues in bold are conserved between E.coli and MMLV, and the secondary structure of E.coli RNase HI from the crystal structure (Katayanagi et al., 1990Go; Yang et al., 1990Go) is indicated. (b) Conserved active site residues and hypothetical position of divalent metal ions are drawn schematically based upon the E.coli and HIV RNase H crystal structures (Katayanagi et al., 1990Go; Yang et al., 1990Go; Davies et al., 1991Go). The location of the lower metal ion (Site I) is based upon the Mg2+ in the E.coli structure and the Mn2+ in the HIV structure, that of the upper metal ion (Site II) is based upon the second Mn2+ observed in HIV RNase H.

 
RNase H can utilize either Mg2+ or Mn2+ for catalysis. Five conserved residues (three aspartates, a glutamate and a histidine) form the metal-binding active site of RNase H (Figure 1bGo). The side chains of these amino acids map to similar positions in the crystal structures of RNases H from E.coli (Katayanagi et al., 1990Go; Yang et al., 1990Go), Thermus thermophilus (Ishikawa et al., 1993bGo) and HIV (Davies et al., 1991Go). In these crystals, E.coli RNase HI binds a single Mg2+ ion via three carboxylates (D10, E48 and D70) that form Site I (Katayanagi et al., 1990Go, 1993bGo), while the HIV RNase H domain binds two Mn2+ ions: one in an analogous position to Site I and another liganded by the C-terminal aspartate (Site II) (Davies et al., 1991Go).

Despite the importance of RNase H to retroviral diseases such as AIDS, most enzymological research on this family of proteins has been conducted on the more simple E.coli homolog. In E.coli RNase HI, conservative mutation of the residues around Site I eliminates essentially all Mg2+-dependent activity (Kanaya et al., 1990Go) while mutations in the histidine and aspartate proximal to Site II (Oda et al., 1993Go; Haruki et al., 1994Go) have lesser effects on the catalytic rates. Based upon these and other studies, several mechanisms (both one-metal and two-metal) have been proposed for RNase H (Davies et al., 1991Go; Nakamura et al., 1991Go; Oda et al., 1993Go; Kanaya et al., 1996Go; Kashiwagi et al., 1996Go; Keck et al., 1998Go) often with the implicit assumption that the catalytic mechanism in the presence of either metal (Mg2+ or Mn2+) is the same. Numerous metal-binding studies have demonstrated that a single Mg2+ ion binds E.coli RNase HI, and, hence, the leading catalytic models for its activity utilize a one-metal mechanism (Katayanagi et al., 1990Go; Oda et al., 1991Go; Katayanagi et al., 1993bGo; Black and Cowan, 1994Go; Huang and Cowan, 1994Go; Kanaya et al., 1996Go). However, several studies have shown that Mg2+-dependent activity is often lost in RNase H variants retaining Mn2+-dependent activity (Stahl et al., 1994Go; Keck and Marqusee, 1995Go; Blain and Goff, 1996Go; Keck and Marqusee, 1996Go; Goedken et al., 1997Go). This raises the possibility of differential binding and/or catalytic uses of these metals by RNase H.

Because the activity from MMLV, both in the full-length RT and the isolated RNase H domain, is greater in the presence of Mn2+ than in Mg2+ (Blain and Goff, 1996Go; Schultz and Champoux, 1996Go; Zhan and Crouch, 1997Go), we investigated the metal binding and activation of MMLV RNase H. Thermal denaturation studies suggest that the aspartates near Site I of MMLV RNase H, but not the glutamate, are important for metal-binding to the enzyme. Mutations that inactivate E.coli RNase HI in the presence of Mg2+ also result in losses in the Mn2+-dependent activity of MMLV RNase H. This suggests a similar one-metal catalytic mechanism for both Mn2+- and Mg2+-dependent RNase H activities and for both prokaryotic and retroviral ribonucleases H.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Creation of MMLV RNase H active site mutants

In order to identify the conserved residues located in the MMLV RNase H active site (Katayanagi et al., 1990Go; Yang et al., 1990Go; Davies et al., 1991Go; Katayanagi et al., 1993bGo) (Figure 1Go), we used sequence alignments of MMLV RT to other RNases H (Johnson et al., 1986Go). Site-directed Kunkel mutagenesis (Kunkel, 1985Go) was used to create a series of point mutations in a plasmid (pEG200) carrying a synthetic gene directing the expression of the C-terminal 175 amino acids of MMLV reverse transcriptase (MRH-175). MRH-175 contains a short extension past the region homologous to E.coli RNase HI which is necessary to produce protein which is fully folded and active (Goedken and Marqusee, 1998Go). DNA sequencing confirmed the incorporation of the desired mutations. Plasmids pEG210, pEG211, pEG212, pEG213 and pEG214 correspond to expression constructs that encode D524N, E562Q, D583N, D653N and H638G MMLV RNase H, respectively. In this report, residue numbers correspond to those from full-length MMLV RT (Copeland et al., 1985Go). MMLV RNase H proteins were expressed and purified as described previously (Goedken and Marqusee, 1998Go).

RNase H activity assays

Radiolabeled RNA·DNA hybrid was synthesized from M13K07 single-stranded DNA, and RNase H assays were conducted as described previously (Keck and Marqusee, 1995Go). Unless otherwise specified, RNase H reaction assays were carried out in standard conditions of 50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1–2 µM (base pairs) RNA·DNA hybrid, 1 mM divalent cation (MnCl2 or MgCl2), 1 mM DTT, 2.5% glycerol, 0.1 mg/ml linear polyacrylamide, 1.5 µM bovine serum albumin (BSA) at 37°C. The acid-soluble radioactivity (cleaved product) present in the supernatant was determined by liquid scintillation counting. Least-square Michaelis–Menten analyses of substrate concentration versus initial reaction velocity were performed using LEONORA Version 1.0.

Circular dichroism measurements

Circular dichroism (CD) data were collected on an Aviv 62DS spectropolarimeter with a Peltier temperature-controlled sample holder and 1-cm pathlength cuvette. Thermal denaturation studies were conducted by monitoring the ellipticity at 227 nm as a function of temperature. Guanidium chloride (GdmCl) was included in thermal denaturation experiments to prevent aggregation and insure >90% reversibility. Midpoint temperatures (Tms) were calculated as described previously (Dabora and Marqusee, 1994Go) using KaleidaGraph (Abelbeck/Synergy Software).


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Design and expression of MMLV RNase H active site mutants

MMLV RT residues D524, E562, D583, H638 and D653 correspond to side chains known to contribute to the active site in E.coli RNase HI (D10, E48, D70, H124, D134) (Figure 1Go). Using site-directed mutagenesis, we introduced conservative mutations in the active site carboxylates (D524N, E562Q, D583N, D653N) and a neutralizing mutation in the conserved histidine (H638G). These mutant proteins were overexpressed and purified to homogeneity as described previously (Goedken and Marqusee, 1998Go). Electrospray-ionization mass spectrometry confirmed the expected molecular weight of all proteins (data not shown).

RNase H activity is decreased in all mutants

The MMLV RNase H mutants were assayed for RNase H activity in the presence of Mn2+. The mutants all showed substantially decreased activity relative to wild-type RNase H (Table IGo): the proteins with mutations that cluster around Site I (D524N, E562Q and D583N) showed very little activity, and the H638G and D653N mutants gave detectable but reduced activity. Therefore, as with E.coli RNase HI, the MMLV RNase H active site residues proximal to Site II appear less important for catalysis than the side chains that cluster around the first metal-binding site. Michaelis–Menten analysis suggested that, in addition to a loss in catalytic rate, H638G has reduced substrate affinity, while D653N shows a similar affinity compared with wild type (data not shown). In 1 mM MgCl2, wild-type MMLV RNase H activity is ~200-fold lower than in the presence of 1 mM MnCl2 (Goedken and Marqusee, 1998Go), and the mutants showed reductions in activity in the presence of Mg2+ roughly similar to those observed in the presence of Mn2+ (data not shown).


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Table I. Catalytic properties of MMLV RNase H variants
 
Metal dependence of MMLV RNase H activity

We probed the concentration requirements for Mn2+ in RNase H catalysis by titrating various amounts of divalent metal into a standard RNase H reaction (Figure 2aGo, Table IGo). MMLV RNase H was increasingly activated by MnCl2 from concentrations of 0.1 to 1 mM and was subsequently inhibited at concentrations greater than 1 mM. A titration of MMLV RNase H with MgCl2 showed a similarly-shaped curve to that obtained in Mn2+ with maximal activity at 1 mM Mg2+ (data not shown) but required ~200-fold more enzyme for equivalent product release. Therefore, the substantial reduction in activity in the presence of Mg2+ relative to Mn2+ for MMLV RNase H cannot simply be the result of weakened binding affinity and must result from inherent differences in the manner these cations contribute to catalysis. We also determined the Mn2+-dependence of H638G and D653N, which have mutations that cluster near metal Site II (Figure 2bGo, Table IGo). Despite reduced specific activities, both proteins showed metal dependence similar to that of the wild-type protein having maximum activity at 1 mM MnCl2 suggesting that the metal-binding affinity of these mutants is not substantially reduced and cannot account for their reductions in activity. In order to separate general ionic effects from specific effects upon the activity of wild-type MMLV RNase H, we titrated NaCl into the assay in the presence of 1 mM Mn2+. NaCl strongly inhibited activity even at concentrations under 100 mM (data not shown) suggesting that a significant amount of the inhibition seen at divalent metal concentrations greater than 1 mM is the result of nonspecific or ionic-strength changes.




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Fig. 2. Metal dependence of MMLV RNase H activity. Reactions were incubated for 10 min at 37°C in standard buffer conditions containing 1 µM base pairs RNA·DNA hybrid. Error bars represent the standard deviation of at least two separate reactions. (a) MnCl2 titration of 5 nM wild type MMLV RNase H (closed circles) and no enzyme control (open diamonds). (b) MnCl2 titration of 50 nM H638G (closed squares) or 50 nM D653N (open circles).

 
Circular dichroism, thermal denaturation and metal binding

All mutant proteins showed circular dichroism (CD) spectra having strong {alpha}-helical character which were superimposable with that of the isolated MMLV RNase H domain (Figure 3Go). Hence, no gross structural rearrangements resulted from the mutations introduced into the active site of MMLV RNase H.



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Fig. 3. Far-UV circular dichroism spectra of MMLV RNase H proteins. Wild type (closed diamonds), D524N (closed circles), E562Q (closed squares), D583N (open squares), H638G (open circles) and D653N (open diamonds). Data were collected using a 1 cm path-length cuvette at 25°C in 5 mM potassium fluoride, 2 mM potassium phosphate, pH 8.0 with 25 µg/ml protein.

 
Metal-binding in MMLV RNase H was evaluated via its linkage to protein stability by carrying out thermal denaturation studies in the presence of various divalent cations (Figure 4Go, Table IIGo). In the absence of metal, wild-type MMLV RNase H displays a cooperative unfolding transition and weak cold denaturation (Goedken and Marqusee, 1998Go). The enzyme was significantly stabilized in the presence of Mn2+ such that its midpoint temperature (Tm) shifted by more than 12°C. Addition of Mg2+ increased the melting temperature to a lesser degree (implying a weaker affinity for this metal), whereas addition of NaCl did not alter the Tm suggesting that the stabilization of MMLV RNase H by divalent metals stems from specific ligand binding.



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Fig. 4. Thermal unfolding of MMLV RNase H in the presence (open circles) and absence (closed circles) of 1 mM MnCl2. Data were collected by monitoring the CD signal at 227 nm in 16.7 mM HEPES pH 8.0, 0.8 M GdmCl with 25 µg/ml protein.

 

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Table II. Thermal denaturation of MMLV RNase H variants in the presence of metal ions
 
In the absence of added metal, all of the active-site mutations except H638G resulted in a higher Tm than the wild-type. The RNase H active site carries four carboxylates in close proximity, and stabilization via mutation of these residues likely results from relief of negative-charge repulsion. Similar effects have been reported for E.coli RNase HI (Kanaya et al., 1996Go). The thermal denaturation profile of H638G was coincident with that of wild type MMLV RNase H despite insertion of a destabilizing glycine residue. This may be because the loop in which this histidine resides is highly flexible as has been observed in RNases H from HIV and E.coli (Katayanagi et al., 1990Go; Yang et al., 1990Go; Davies et al., 1991Go; Powers et al., 1991Go).

Metal-dependent thermostabilization offers a way to assess protein–metal interactions that does not require enzymatically-active protein. Partially-active mutants H638G and D653N showed melting temperature shifts in both Mn2+ and Mg2+ (Figure 4Go, Table IIGo). The inactive mutants D524N and D583N showed virtually identical Tms with and without metal. However, the inactive mutant E562Q showed large stabilization with the inclusion of Mn2+ or Mg2+. This implies that the inactivity of D524N and D583N is due to an inability to bind metal needed for catalysis while the inactivity of E562Q results from a more subtle effect upon the enzyme.


    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
RNase H activity is crucial for retroviral replication (Tisdale et al., 1991Go), but the enzymatic mechanism of the structurally-similar RNase HI protein from E.coli has been studied more thoroughly than its retroviral homologs. This is, in part, because prokaryotic RNases H lack the large N-terminal polymerase domains of RNase H-containing retroviral RTs which complicate mechanistic analyses. Mutational studies in the E.coli protein, carried out largely in the presence of Mg2+, have led to several models for its catalytic mechanism (Oda et al., 1993Go; Haruki et al., 1994Go; Kanaya et al., 1996Go; Kashiwagi et al., 1996Go), but whether these models apply to the entire RNase H family in either Mn2+ or Mg2+ is unknown. Interestingly, numerous mutations in E.coli and HIV RNase H result in enzymes active in the presence of Mn2+ but inactive in Mg2+ (Evans et al., 1991Go; Smith and Roth, 1993Go; Stahl et al., 1994Go; Keck and Marqusee, 1995Go; Keck and Marqusee, 1996Go; Goedken et al., 1997Go). Furthermore, E.coli RNase HI and HIV RT are more active in the presence of Mg2+ than Mn2+, whereas MMLV RT shows greater RNase H activity in Mn2+ (Blain and Goff, 1996Go), suggesting intriguing differences in the ways these metals activate different enzymes. Finally, the crystal structures of HIV RNase H show two Mn2+ ions in the active site (Davies et al., 1991Go), whereas E.coli RNase HI binds only one Mg2+ ion as judged by several methods (Katayanagi et al., 1990Go; Oda et al., 1991Go; Katayanagi et al., 1993bGo; Black and Cowan, 1994Go; Huang and Cowan, 1994Go), raising the question of whether one or two metal ions are needed for catalysis.

The MMLV RNase H domain serves as a model retroviral ribonuclease H, and because Mn2+ is the optimal metal for this RT, we investigated whether the residues necessary for catalysis in E.coli RNase HI were also required for activity and metal binding in the context of the MMLV enzyme. Our results strengthen the notion that two metal ions are not required in the MMLV RNase H active site for catalysis and that Mn2+-dependent RNase H activity shares a common mechanism with that using Mg2+ ions.

Site I metal binding

Though high-resolution data of the MMLV RNase H active site is not yet available, given that E.coli RNase HI and the MMLV and HIV RNase H domains all share ~25% sequence identity, we expect that the active site of MMLV RNase H resembles that from its homologs (Figure 1Go). In the crystal structure of E.coli RNase HI, a Mg2+ ion was observed near the side chains of D10, E48 and D70 at distances of 2.1, 2.4 and 4.4 Å respectively (Katayanagi et al., 1990Go; Katayanagi et al., 1993bGo) whereas in the HIV RNase H domain, a Mn2+ ion was located 2.8, 1.9 and 4.1 Å respectively from the homologous residues (Davies et al., 1991Go). We refer to this position as Site I. Single mutations in these residues in E.coli (D10N, E48Q, D70N) almost completely inactivate the enzyme, and these three variant proteins show minimal deviations from the wild-type structure by X-ray crystallography (Katayanagi et al., 1993aGo). Here, we show similar decreases in Mn2+-dependent activity via conservative mutations (D524N, E562Q, D583N) in the isolated MMLV RNase H domain suggesting these active-site residues are necessary for catalysis in the presence of either divalent metal. This is significant because, despite the many discrepancies in how Mn2+ and Mg2+ ions affect RNases H, both metals share the same basic requirements for Site I proximal residues.

What, then, are the specific roles of these Site I carboxylates in catalysis? In E.coli RNase HI, the position of a single Mg2+ ion near D10, E48 and D70 (Katayanagi et al., 1990Go; Katayanagi et al., 1993bGo) suggests that at least some of these side chains are responsible for metal-ion affinity. Earlier one-metal mechanistic proposals suggested D70 functions as a general base activating water by producing a hydroxide ion competent to attack the scissile RNA phosphodiester bond and that D10 and E48 position the divalent metal (Oda et al., 1993Go; Katayanagi et al., 1993bGo). This divalent cation may stabilize excess negative charge on the pentacovalent phosphorane intermediate to enable catalysis. Mg2+-binding to active site mutants D10N and D70N is greatly impaired whereas that for E48Q is largely unchanged leading to the proposal that rather than a metal-binding element, E48 functions to align and later eject a water molecule which acts as a general acid (Kanaya et al., 1996Go). Kanaya et al. (1996) further proposed that RNA hydrolysis relies upon D10 and D70 to align the divalent metal and H124 as a water-activating general base.

Our thermal denaturation study of MMLV RNase H also suggests that D524 and D583 (the analogs of E.coli D10 and D70) are more crucial for binding of divalent metal than E562. Therefore, despite the close proximity of the metal ions in the E.coli and HIV crystal structures to the active-site glutamate, metal-binding studies in two highly-diverged RNases H suggest that this conserved residue is not necessary for metal binding. The apparent discrepancies between seemingly-contradictory crystallographic and metal-binding studies in this enzyme family are not easily resolved. Perhaps the energetics of the interaction between the glutamate and the divalent ion are not as favorable as one might imagine from their position in the crystal structure. If the active-site glutamate is not important for anchoring a metal ion, why then, is it crucial for catalysis? Perhaps it does anchor a water molecule needed for general-acid catalysis (with histidine as a general base) as in the model of Kanaya et al. (1996). Alternatively, the glutamate could act as a general base producing the required nucleophilic hydroxide or play an essential role in orienting the enzyme–substrate complex for catalysis.

Site II metal binding

MMLV RNase H residues D653 and H638 cluster near its putative second metal-binding site. In the isolated HIV RNase H domain the homologous side chain to D653 is 2.0 Å from the second Mn2+ ion observed in metal-soaked crystals (Davies et al., 1991Go). The loop containing the conserved histidine is disordered in this structure and although this region of the protein is likely highly flexible, the histidine side chain is located near the putative second-metal (Site II) in several other RNase H structures (Yang et al., 1990Go; Kohlstaedt et al., 1992Go; Ishikawa et al., 1993aGo; Katayanagi et al., 1993aGo; Kashiwagi et al., 1996Go). MMLV Site II mutants D653N and H638G mutants have modest (7- and 25-fold) reductions in activity, suggesting that while these residues are not as crucial as D524, E562 and D583, they are significant for catalysis. Similar mutations in E.coli RNase HI (D134N and H124A) have 90% and ~2% wild-type activity respectively in Mg2+ (Kanaya et al., 1990Go).

The activity losses seen from mutations in D653 and its homologs likely stem from subtle electrostatic and conformational changes in the active site and not because a metal ion at Site II is necessary for catalysis. If two-metals were required for RNase H catalysis, we would expect that the affinity for the metal in Site II of D653N would be drastically reduced, and that this would be reflected in the metal dependence of activity. Instead, the metal-dependence curve of D653N is similar to that of the wild-type (Figure 2Go) with maximum activity at 1 mM, despite an overall reduction in activity. Therefore, unless a second metal liganded by D653 is needed only to increase activity 7-fold (to wild-type levels), our results support a one-metal mechanism for MMLV RNase H rather than a mechanism requiring two metal ions. This idea is further supported by the observation that mutations in Site I (D524N and D583N) appear to greatly limit metal binding in the thermal denaturation studies while those in Site II (D653N and H638G) continue to bind metal (Figure 4Go, Table IIGo).

Interestingly, E.coli RNase HI is activated and subsequently strongly inhibited by Mn2+ at low concentrations (~5 µM) (Keck and Marqusee, 1996Go). These data suggest an `activation/attenuation' model for E.coli RNase HI where a metal of higher affinity (Site I) activates catalysis and a second, lower affinity metal (Site II) inhibits activity (Keck et al., 1998Go). Hence, while two metal ions can bind RNase H, only one is necessary for catalysis. In MMLV RNase H, however, the mutations proximal to Site II (D653N and H638G) do not alter the inhibitory regime of Mn2+ previously observed for the wild-type protein (Figure 2Go). Because it can be mimicked by NaCl, this Mn2+-inhibition phase of MMLV RNase H is likely due primarily to nonspecific effects of metal in the reaction. Hence, unlike E.coli RNase HI, the isolated MMLV RNase H domain does not appear to function in an attenuation mode. Given the importance of Site I as discussed above, we therefore propose that in MMLV RNase H, a single, activating Mn2+ ion binds in Site I and that Site II is not significantly occupied.

Implications for full-length RT

We have demonstrated that the metal activation and binding properties of isolated MMLV RNase H are similar to those of E.coli RNase HI which appears to use a single divalent metal ion for catalysis. It has been previously shown that mutations in D524 and D583 of full-length MMLV RT greatly reduce its RNase H activity and viral infectivity (Repaske et al., 1989Go; Blain and Goff, 1993Go). Furthermore, mutations in the homologs of D653 and H638 in HIV RT have lesser effects on RNase H activity (Schatz et al., 1989Go; Wohrl et al., 1991Go; Rausch and Le Grice, 1997Go). Hence, the results of our studies on the isolated RNase H domain are likely pertinent to full-length reverse transcriptases, the relevant target for anti-retroviral drug design.

In addition to general removal of genomic RNA, however, specific RNase H-cleavage events (the removal of the tRNA primer and creation of the polypurine tract) are required for reverse transcriptase to perform its biological function (reviewed in Champoux, 1993Go; Hottiger and Hubscher, 1996Go). While the MMLV RNase H domain is active when removed from the polymerase portions of RT, it does not retain the specificity of the full-length protein for these substrates (Schultz and Champoux, 1996Go; Zhan and Crouch, 1997Go), suggesting substrate discrimination is directed in an unknown manner by the polymerase domain. Therefore, additional work is needed to compare and contrast the mechanisms of these two modes of cleavage in RNase H.


    Acknowledgments
 
We thank David King for mass spectrometry, James Keck, Andrew Mesecar, Srebrenka Robic and Julie Hollien for critical reading of this manuscript, and the members of the Marqusee Laboratory for helpful discussions. This work was supported by a grant from the NIH (GM53321).


    Notes
 
1 To whom correspondence should be addressed;email: marqusee{at}zebra.berkeley.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received March 5, 1999; revised June 5, 1999; accepted July 16, 1999.