Department of Molecular and Cell Biology, University of California, Berkeley, 229 Stanley Hall, Berkeley, CA 94720, USA
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Abstract |
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Keywords: divalent metals/nucleic acid hydrolysis/retrovirus/RNase H/reverse transcriptase
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Introduction |
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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., 1990) while mutations in the histidine and aspartate proximal to Site II (Oda et al., 1993
; Haruki et al., 1994
) 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., 1991
; Nakamura et al., 1991
; Oda et al., 1993
; Kanaya et al., 1996
; Kashiwagi et al., 1996
; Keck et al., 1998
) 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., 1990
; Oda et al., 1991
; Katayanagi et al., 1993b
; Black and Cowan, 1994
; Huang and Cowan, 1994
; Kanaya et al., 1996
). However, several studies have shown that Mg2+-dependent activity is often lost in RNase H variants retaining Mn2+-dependent activity (Stahl et al., 1994
; Keck and Marqusee, 1995
; Blain and Goff, 1996
; Keck and Marqusee, 1996
; Goedken et al., 1997
). 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, 1996; Schultz and Champoux, 1996
; Zhan and Crouch, 1997
), 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.
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Materials and methods |
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In order to identify the conserved residues located in the MMLV RNase H active site (Katayanagi et al., 1990; Yang et al., 1990
; Davies et al., 1991
; Katayanagi et al., 1993b
) (Figure 1
), we used sequence alignments of MMLV RT to other RNases H (Johnson et al., 1986
). Site-directed Kunkel mutagenesis (Kunkel, 1985
) 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, 1998
). 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., 1985
). MMLV RNase H proteins were expressed and purified as described previously (Goedken and Marqusee, 1998
).
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, 1995). Unless otherwise specified, RNase H reaction assays were carried out in standard conditions of 50 mM TrisHCl, pH 8.0, 50 mM NaCl, 12 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 MichaelisMenten 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, 1994) using KaleidaGraph (Abelbeck/Synergy Software).
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Results |
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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 1). 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, 1998
). 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 I): 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. MichaelisMenten 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, 1998
), 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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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 2a, Table I
). 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 2b
, Table I
). 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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All mutant proteins showed circular dichroism (CD) spectra having strong -helical character which were superimposable with that of the isolated MMLV RNase H domain (Figure 3
). Hence, no gross structural rearrangements resulted from the mutations introduced into the active site of MMLV RNase H.
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Metal-dependent thermostabilization offers a way to assess proteinmetal interactions that does not require enzymatically-active protein. Partially-active mutants H638G and D653N showed melting temperature shifts in both Mn2+ and Mg2+ (Figure 4, Table II
). 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.
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Discussion |
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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 1). 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., 1990
; Katayanagi et al., 1993b
) 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., 1991
). 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., 1993a
). 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., 1990; Katayanagi et al., 1993b
) 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., 1993
; Katayanagi et al., 1993b
). 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., 1996
). 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 enzymesubstrate 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., 1991). 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., 1990
; Kohlstaedt et al., 1992
; Ishikawa et al., 1993a
; Katayanagi et al., 1993a
; Kashiwagi et al., 1996
). 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., 1990
).
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 2) 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 4
, Table II
).
Interestingly, E.coli RNase HI is activated and subsequently strongly inhibited by Mn2+ at low concentrations (~5 µM) (Keck and Marqusee, 1996). 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., 1998
). 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 2
). 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., 1989; Blain and Goff, 1993
). Furthermore, mutations in the homologs of D653 and H638 in HIV RT have lesser effects on RNase H activity (Schatz et al., 1989
; Wohrl et al., 1991
; Rausch and Le Grice, 1997
). 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, 1993; Hottiger and Hubscher, 1996
). 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, 1996
; Zhan and Crouch, 1997
), 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.
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Acknowledgments |
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Notes |
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Received March 5, 1999; revised June 5, 1999; accepted July 16, 1999.