Hyperactivity of Human DNase I Variants
DEPENDENCE ON THE NUMBER OF POSITIVELY CHARGED RESIDUES AND CONCENTRATION, LENGTH, AND ENVIRONMENT OF DNA*

Clark Q. Pan and Robert A. LazarusDagger

From the Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human DNase I, an enzyme used to treat cystic fibrosis patients, has been engineered to more effectively degrade double-stranded DNA to lower molecular weight forms by introducing positively charged amino acids at positions that can interact favorably with the proximal negatively charged phosphate groups of the DNA. A series of combination mutants having from one to six additional basic residues compared with the wild type has been constructed, expressed in human 293 cells, and characterized. The degree of hyperactivity for the mutants was highly dependent upon the conditions in various assays, including the concentration and length of the DNA substrate and the salt and divalent metal ion concentrations. The level of hyperactivity was inversely proportional to both DNA concentration and DNA length, consistent with the processive nicking mechanism for the hyperactive variants. Salt was inhibitory for wild type DNase I but actually enhanced the activity of the hyperactive variants. Under optimal conditions for wild type, variants with one additional positive charge possessed the highest activity, which was only severalfold greater than that for wild type. However, in the presence of low DNA concentrations and molecular weights, no Ca2+, and 150 mM NaCl, the variant with six engineered basic residues was most active, having >10,000-fold higher activity than the wild type enzyme. Therefore, any potential increase in potency for the hyperactive variants in vivo will be determined by the concentration, length, and environment of the DNA.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recombinant human DNase I is an important clinical agent that is currently used for the treatment of cystic fibrosis patients (1). It is inhaled into the airways, where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of cystic fibrosis sputum and improving lung function (2, 3). In addition, the use of DNase I in a murine model of systemic lupus erythematosus has been investigated with encouraging results (4). The pharmacological and clinical significance of DNase I has led us to recently engineer actin-resistant variants of the human enzyme that are no longer inhibited by actin (5), as well as hyperactive variants that alter the functional mechanism of DNA cleavage (6); characterization of these variants under biologically relevant ex vivo conditions has shown a marked improvement in potency compared with wild type.1 Furthermore, we have recently addressed the functional importance of various residues of human DNase I at the DNA binding interface by extensive mutagenesis (7).

DNase I is an endonuclease that catalyzes the hydrolysis of double-stranded DNA predominantly by a single-stranded nicking mechanism under physiological conditions when both Ca2+ and Mg2+ ions are present (8). The hyperactive variants utilized a more efficient functional mechanism involving processive nicking, which yielded a greater number of double-stranded breaks as a result of higher affinity for DNA (6). Eight "hyperactive" positions were selected from a mutational scanning analysis of the DNase I-DNA interface, where Arg or Lys replaced the native residues (Fig. 1). The engineered positive charges were designed to generate attractive interactions with the negatively charged phosphates on the DNA backbone. In addition, the tighter binding to DNA also eliminated the significant inhibition of DNase I by physiological saline. The combination of three hyperactive positions led to a +3 variant2 with three additional positive charged residues and ~35-fold higher DNA cutting activity relative to wild type. In the present study, we have constructed seven new combination mutants that have up to six more positive charges than the wild type. These have been expressed in human 293 cells to determine the degree and limits of hyperactivity under an array of different assay conditions in which the concentration and length of the DNA substrate, the salt concentration, and the type of metal ions were varied. Under selected conditions, the hyperactive variant that has six additional positive charged residues was >10,000-fold more active than the wild type enzyme.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Molecular Modeling-- All modeling work was performed with the InsightII program (Molecular Simulation, Inc., San Diego). The crystal structure of human DNase I (9) was superimposed onto that of the bovine DNase I-octamer complex (10); the backbone root mean square deviation was 0.5 Å. The six hyperactive positions that were combined into the +6 variant were replaced with either lysine or arginine (Fig. 1A). The orientation of each introduced basic amino acid side chain was selected from a rotamer library (11) based on three criteria. The chosen rotamer had one of the lowest energies, had no steric clashes with DNA or the rest of the protein, and placed the side chain amino group near the DNA phosphate oxygens. With the exception of residue 9, the nitrogen stemming from each engineered Arg or Lys was between 3 and 4 Å from a phosphate oxygen. The direction of Arg9 modeled in the +6 human variant was similar to those found in the two structures of DNA complexed to bovine DNase I (10, 12), which has an arginine at position 9. The electrostatic potentials of the wild type human DNase I and its +6 variant (Fig. 1B) were calculated by using the default setups of the Delphi program (Molecular Simulation, Inc., San Diego, CA).

Mutagenesis, Expression, and Characterization-- Site-directed mutagenesis, DNase I expression in human 293 cells and secretion into the culture media, human DNase I ELISA, and actin binding ELISA measurements were carried out essentially as described previously (5, 6). Multiple mutations were introduced into DNase I by including several single mutant primers in one hybridization reaction (13). The actin binding affinity of all of the hyperactive variants described herein was the same as wild type, implying that their overall structural integrity remained intact. Those variants with the A114F mutation no longer bound to actin, as predicted based upon previous mutational data at this position (5). All characterization assays were carried out in 25 mM Hepes, pH 7. Mg2+ and Ca2+ concentrations ranged from 1 to 2.5 mM; no significant differences were observed in this concentration range. Physiological concentrations of 1 mM Mg2+ and 2.5 mM Ca2+ were used in most experiments.

DNA-Methyl Green Assay-- The methyl green assay was used to measure DNA hydrolytic activity of DNase I in the presence of 2 mM Mg2+ and 2 mM Ca2+ as described previously (14). Salmon testes DNA (Sigma), up to ~25,000 bp3 in size, was mixed with methyl green (Sigma) to create the DNA-methyl green substrate with a final DNA concentration of 770 µg/ml, used for the assay. DNase I concentrations were determined by enzyme-linked immunosorbent assay using a goat anti-DNase I polyclonal antibody coat and detecting with a rabbit anti-DNase I polyclonal antibody conjugated to horseradish peroxidase. In both assays, multiple sample dilutions were compared with standard curves of wild type DNase I to determine concentrations.

Kinetic Measurements-- Kinetic assays were based on the Kunitz hyperchromicity assay (15). Diluted culture media was incubated with 10-300 µg/ml calf thymus DNA (Sigma) in 25 mM Hepes, pH 7, 1 mM MgCl2, 2.5 mM CaCl2, and 150 mM NaCl at room temperature. The subsequent increase in absorbance at 260 nm was monitored with a Molecular Devices Spectra Max 250 spectrophotometer. No activity was detected in mock-transfected media. Plots of initial rates of A260 increase versus DNA concentration were hyperbolic for most variants, and the data were fit by nonlinear regression analysis to the Michaelis-Menten equation to generate apparent Km and Vmax kinetic values. For the salt inhibition assay (see Fig. 6A), the DNA concentration was fixed at the saturating concentration of 300 µg/ml, and the amount of NaCl was varied from 0 to 400 mM.

Plasmid Digestion Assay in Native Agarose Gel-- Supercoiled pBR322 plasmid DNA (New England Biolabs) at 29 µg/ml or linearized pBR322 (EcoRI-digested and purified by phenol-chloroform extraction and ethanol precipitation) at 25 µg/ml was treated with diluted culture media from human 293 cells transfected with wild type or variant DNase I in the presence of 25 mM Hepes, pH 7, 100 µg/ml bovine serum albumin, 1 mM MgCl2, 2.5 mM CaCl2 with varying NaCl concentrations at room temperature. At various time intervals, aliquots of the reaction mix were quenched with 25 mM EDTA, 6% glycerol, xylene cyanol, and bromphenol blue and loaded directly on a 0.8% agarose gel. The gel was run overnight at ~1 V/cm in TBE and stained with ethidium bromide, and individual bands were quantified with a Molecular Dynamics model 575 FluorImager. The overall activity was measured as the initial rate of disappearance of supercoiled or linear substrate. The linear to relaxed ratio was also determined from the initial rates of appearance of linear and relaxed products. Mock-transfected media showed no background activity.

32P-Labeled DNA Digestion-- EcoRI-linearized plasmid pBR322 (4361-mer) was radiolabeled at the 3'-end using [alpha -32P]dATP (Amersham Pharmacia Biotech) and the Klenow fragment of DNA polymerase I (U. S. Biochemical Corp.) (16). A fraction of the labeled pBR322 was then digested separately with HindIII (New England Biolabs) and PstI (Amersham Pharmacia Biotech), followed by polyacrylamide gel purification of the respective 32-mer and 752-mer restriction fragments. Radiolabeling of the 190-mer EcoRI-SspI fragment of pBR322 has been described previously (6). 32P-Labeled linearized pBR322 at concentrations ranging from 8.6 ng/ml (3 pM) to 29 µg/ml (10 nM), the 190-mer fragment at 85 ng/ml (680 pM), and the 752-mer fragment at 6.3 ng/ml (12.7 pM) were treated with diluted culture media from DNase I-transfected 293 cells at room temperature in the presence of 25 mM Hepes, pH 7, 1 mM MgCl2, 1 mM CaCl2, and 100 µg/ml bovine serum albumin. For the 4361-mer and 752-mer fragments, the reaction was quenched with 25 mM EDTA, 6% glycerol, 50 mM NaOH, xylene cyanol, and bromphenol blue and loaded onto a 0.8% denaturing agarose gel in 50 mM NaOH. For the 190-mer fragment, the reaction was stopped with 10 mM EDTA, xylene cyanol, and bromphenol blue in deionized formamide and run on a 6% denaturing polyacrylamide gel. The labeled 32-mer fragment at 6.3 ng/ml (300 pM) was treated with DNase I in the presence of 25 mM Hepes, pH 7, 1 mM MgCl2, 1 mM CaCl2, 150 mM NaCl, and 100 µg/ml bovine serum albumin; in some reactions, CaCl2 was replaced by 0.1 mM EGTA and NaCl was omitted. Reactions were stopped with 10 mM EDTA, xylene cyanol, and bromphenol blue in deionized formamide and run on a 20% denaturing polyacrylamide gel. The gels were dried and incubated with a Fuji phosphorimaging plate (type BAS-III), which was then scanned with a Fuji phosphorimager (BAS2000). The activity was measured as the initial rate of disappearance of linear DNA substrate.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of Combination Hyperactive DNase I Variants-- Eight hyperactive positions of DNase I were previously identified using a basic residue scan of the DNase I-DNA interface (6). Based upon data from two different combination variants at some of these positions, the degree of hyperactivity reflected the additivity of individual mutations, at least under a given set of conditions. In the present study, we constructed, expressed, and characterized multiple variants having from one to six additional positively charged amino acids at these hyperactive positions (Fig. 1A) to test the effects of progressively increasing the number of positive charges on DNase I activities. These engineered basic residues altered the electrostatic surface at the DNA binding interface such that a positive potential was created at areas that are distal to and distinct from the active site (Fig. 1B). The degrees of hyperactivity of these variants were assessed in a series of assays under different conditions. In particular, the DNA concentration ranged from ng/ml to mg/ml, and the DNA length varied from 32 to ~25,000 bp. In addition, the effects of salt and divalent metal ion concentrations were also determined for these variants.


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Fig. 1.   Model of the +6 hyperactive human DNase I-DNA complex. A, six hyperactive positions at the DNA binding interface of the human DNase I-d(GGTATACC)2 complex are labeled and colored in light blue. The model is derived from superposition of human DNase I (9) with the bovine DNase I-octamer complex (10) and substituting each of the six hyperactive positions with lysine or arginine as found in the +6 variant. The orientations of the introduced basic amino acids were chosen from one of the seven rotamers found in a rotamer library (11). The possible attractive interactions between the positively charged amino groups of these engineered residues with the negatively charged phosphate oxygens (red) of the DNA (green) are highlighted by pink dashes. B, electrostatic potential calculations of wild type DNase I (left) and +6 variant DNase I (right). The view of DNA (green) complexed to DNase I in A is rotated approximately 90°. The positive potential is colored in blue, whereas the negative potential is in red, with a scale of ± 5 kT/e.

DNA-Methyl Green Assay-- Hyperactive variants were first tested in the DNA-methyl green assay, which measures a decrease of A620 as the methyl green dye is released from hydrolyzed DNA. This assay was used to determine the hydrolytic activities of hyperactive DNase I variants in the presence of 770 µg/ml DNA that is up to ~25,000 bp in length, Mg2+, Ca2+, and no NaCl (14). Single basic residue replacements showed relatively modest increases in activity relative to wild type (Table I). The addition of more positive charges progressively reduced the degree of hyperactivity to the extent that variants with five or six more charges were actually less active than wild type.

                              
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Table I
Relative DNA digestion activity of human DNase I variants
All experiments were carried out under physiological conditions of 1 mM Mg2+, 2.5 mM Ca2+, and 150 mM NaCl at pH 7, with the exception of the DNA-methyl green assay, which was performed in 2 mM Mg2+ and 2 mM Ca2+. All values are normalized relative to those of wild type, which has a Km of 229 ± 33 µg/ml DNA (n = 6), Vmax of 168 ± 18 A260 units · min-1 · mg-1 DNase I (n = 6), linear DNA cleavage activity of 300 ± 40 pM DNA · min-1 · nM-1 DNase I (n = 6), a linear/relaxed product ratio (L/R) of 0.010 for supercoiled DNA digestion with overall loss of the supercoiled substrate DNA at 15400 ± 550 pM DNA · min-1 · nM-1 DNase I (n = 2), and 1283 ± 34 methyl green units · mg-1 DNase I (n = 40). n denotes the number of separate experiments done. Errors were derived from standard errors of multiple data points except for the hyperchromicity data, which were derived from statistics of the fit. Data for the wildtype, the +1 and +2 variants, and the Q9R/E13R/N74K variant were taken from a previous study and are included for completeness (6).

Kinetic Characterization-- Variants were then tested in the DNA hyperchromicity assay in which the A260, due to the DNA absorption, increases as a function of degradation (15). Plots of initial velocity versus substrate concentration (10-300 µg/ml and up to ~25,000 bp) yielded hyperbolic curves for wild type DNase I and most variants (Fig. 2), allowing the extraction of apparent Km and Vmax values using the Michaelis-Menten equation (Table I). Like the single variants, variants with multiple mutations displayed hyperactivities that can be ascribed to both a decrease in Km, suggesting stronger DNA affinity, and an increase in Vmax. A maximum of 7-8-fold higher Vmax than wild type was generally observed for variants having three or four additional positive charges (Table I). Further increases in the number of positive charges (+5 and +6 mutants) continued to lower the Km but also lowered the Vmax, suggesting that the decrease in turnover number may be a result of binding to the DNA too tightly. Overall, in the presence of Mg2+, Ca2+, 150 mM NaCl, and relatively high DNA concentrations and molecular weights, a maximal improvement in the Vmax/Km of 30-40-fold was found for variants containing three or four additional positive charges relative to wild type.


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Fig. 2.   DNA hyperchromicity assay of human DNase I variants. Calf thymus DNA at the specified DNA concentrations was treated with DNase I variants in the presence of 1 mM Mg2+, 2.5 mM Ca2+, 150 mM NaCl at pH 7; the increase in absorbance at 260 nm as a result of DNA degradation was followed. The initial velocity is plotted against the DNA concentration. The curves were generated by fitting the Km and Vmax values to the Michaelis-Menten equation. Asterisk refers to variant made in actin-resistant A114F background.

Supercoiled Plasmid Digestion Assay-- The degradation of supercoiled plasmid pBR322 to relaxed circle or linearized product permits an assessment of the degree of "nicking" versus "cutting" for different DNase I variants. The lower Km values observed for the hyperactive variants suggest that they bind to DNA tighter and thus may stay on the DNA longer, potentially allowing some double-stranded DNA scission instead of nicking one of the strands as observed for wild type. The introduction of a single positive charge in human DNase I created hyperactive variants that yielded a linear to relaxed product ratio that was 2-5-fold greater than wild type, and the subsequent progressive introduction of additional positively charged residues resulted in an increase in the linear to relaxed product ratio of >200-fold over wild type (Table I). In addition to the number of charges, the specific location of the mutation also affected this ratio, as exemplified by the differences between the two +3 mutants Q9R/E13R/N74K and E13R/N74K/T205K. Furthermore, the +6 variant appeared to yield lower molecular weight products prior to much disappearance of the supercoiled substrate as indicated by the smear running slightly faster than the linear product (Fig. 3), consistent with processive DNA degradation. The maximal overall supercoiled DNA nicking activity of ~7-fold greater than wild type, as measured by the disappearance of the supercoiled substrate, was attained with just one basic amino acid substitution in the N74K variant (Table I). In general, introducing additional basic residues progressively reduced the overall nicking activity with the +6 mutant possessing an activity that was actually less than wild type. These results again support the notion that stronger DNA affinity and a lower turnover number are accompanied by increasing the number of positive charges at the hyperactive positions on DNase I. 


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Fig. 3.   Supercoiled plasmid DNA digestion by human DNase I variants. Supercoiled pBR322 substrate with a low level of relaxed circle background (left lane; C, control) was incubated with DNase I variants in the presence of 1 mM Mg2+, 2.5 mM Ca2+, 150 mM NaCl at pH 7 for 1-45 min. DNase I concentration was 27 pM for wild type (WT) and 2.7 pM for the hyperactive variants. The linear product (L) ran in between the slower moving relaxed product (R) and the faster moving supercoiled substrate (S). The figure was made using Adobe Photoshop, version 3.0.5.

Linear Plasmid DNA Digestion Activity-- The data from the supercoiled plasmid cleavage predict that these positively charged DNase I variants should be more active than wild type in degrading linearized plasmid DNA. Under conditions similar to those of the hyperchromicity assay (Mg2+, Ca2+, 150 mM NaCl, and relatively high DNA concentrations (25 µg/ml) and length (4361 bp)), comparable levels of hyperactivities were observed in the linear DNA digestion assay (Table I). The +3 and +4 variants again possessed the greatest activities, having a 30-40-fold increase relative to wild type. Interestingly, the +6 mutant displayed a linear DNA digestion activity ~7-fold higher than that of the wild type, even though its overall supercoiled DNA nicking activity was only 60% of the wild type activity. This is consistent with earlier data and again implies that it is not the total number, but rather the type of catalytic events, i.e. double-stranded scission, that imparts hyperactivity (6).

DNA Nicking as a Function of DNA Concentration-- Our previous data showed that the +2 variant E13R/N74K was ~20-fold more active than wild type when nicking 0.5 µg/ml of a 190-bp DNA fragment but ~2-fold less active when hydrolyzing 30 µg/ml of a DNA fragment with 4361 bp (6). To resolve the apparent paradox, we have determined the degree of hyperactivity by varying both the DNA concentration and DNA length. Radiolabeled plasmid pBR322 at concentrations ranging from 3 pM (8.6 ng/ml) to 10 nM (29 µg/ml) was treated with different amounts of wild type and the +2 hyperactive variant E13R/N74K in the presence of Mg2+, the presence of Ca2+, and the absence of NaCl (Fig. 4). The mutant had single-stranded nicking activity 19 ± 1 times higher than that of the wild type at low DNA concentrations (10 pM) but 2.5 ± 0.5-fold lower activity at high substrate concentrations (10 nM) (Fig. 4A). Plots of initial rates versus DNA concentrations were hyperbolic for both the native and mutant DNase I and fit the Michaelis-Menten equation well (Fig. 4B). Wild type DNase I and E13R/N74K have Km values of 5.7 ± 0.8 nM (16 ± 2 µg/ml) and 0.11 ± 0.2 nM (0.32 ± 0.05 µg/ml) DNA, respectively, and Vmax values of 183 ± 12 and 51 ± 1 nM DNA·min-1·nM-1 DNase I (14200 ± 900 and 4000 ± 100 mg of DNA·min-1·mg-1 DNase I), respectively. The general trend of the positively charged variants displaying greater hyperactivity at lower DNA concentrations holds true for mutants with one to six additional positive charges (data not shown). For example, the +1 mutant N74K was ~15-fold more active than wild type when digesting 3 pM of pBR322 but only 3-fold more active when the substrate concentration was elevated to 10 nM. Similarly, the +6 variant was ~3-fold more active but ~10-fold less active than wild type at 50 pM and 10 nM pBR322 substrate, respectively.


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Fig. 4.   DNA nicking of the 4361-bp linearized plasmid pBR322 by the wild type human DNase I and the E13R/N74K variant. A, representative denaturing polyacrylamide gels showing DNA digestion by DNase I. The 4361-bp DNA at 0.01-10 nM was treated with 5.4 pM of wild type DNase I or 0.27-10.8 pM of E13R/N74K in the presence of 1 mM Mg2+ and 1 mM Ca2+. The amount of time (0.2-25 min) for DNA digestion is listed at the top of each lane; C, a control DNA that was not incubated with DNase I; WT, wild type. B, the initial rate of disappearance of the 4361-bp DNA substrate is plotted against DNA concentration; several additional concentrations were tested in addition to those shown in A. The data fit the Michaelis-Menten equation well, having r2 values of 0.996 and 0.988 for wild type and E13R/N74K, respectively.

DNA Nicking as a Function of DNA Length-- To address the DNA size dependence of hyperactivity, we determined the DNase I nicking activities of DNA fragments with four different lengths. At a substrate concentration of 6-8 ng/ml, the degree of hyperactivity for the E13R/N74K variant in the presence of Mg2+ and Ca2+ progressively increased from ~20-fold over wild type for the 4361-bp plasmid pBR322 to ~30- and ~200-fold for its 752- and 32-bp restriction fragments, respectively (Table II). Likewise, at a fixed DNA concentration of 85 ng/ml, the level of hyperactivity for the +2 mutant nearly doubled when the DNA size was reduced from 4361 to 190 bp. The amount of improvement in nicking activity for N74K over the wild type also increased significantly with shorter DNA lengths. Therefore, the degree of hyperactivity by DNase I variants with additional positively charged residues at its DNA binding interface is inversely proportional not only to substrate concentration but also to DNA length.

                              
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Table II
Dependence of relative DNA nicking activity by human DNase I variants on DNA length and concentration
Experiments were performed in the presence of 1 mM Mg2+, 1 mM Ca2+, and either the 32-bp EcoRI-HindIII pBR322 fragment, the 190-bp EcoR I-Ssp I fragment, the 752-bp EcoRI-PstI fragment, or the entire 4361-bp linearized pBR322 at various concentrations, listed on the first row. All values are normalized relative to those of the wild type, which has a nicking activity of 56 ± 3 (n = 7), 101 ± 21 (n = 2), 100 ± 8 (n = 3), 470 ± 30 (n = 3), and 970 ± 100 (n = 3) pM DNA · min-1 · nM-1 DNase I for columns 2-6, respectively. n denotes the number of separate experiments performed.

DNA Nicking at Low DNA Concentration and Short DNA Length-- To determine the degree of hyperactivity under optimal conditions for the mutants, we examined their relative nicking activities using a short 32-bp fragment of DNA and at a very low concentration of 6.3 ng/ml under different metal ion and salt conditions (Table III). Under more favorable conditions for wild type, i.e. in the presence of Mg2+, the presence of Ca2+, and the absence of NaCl, the +2 mutant displayed the greatest hyperactivity, being ~200-fold higher than wild type; the addition of two (+4) or four (+6) more positive charges resulted in a dramatic decrease to only 7-fold more active. The absence of Ca2+ destabilized the wild type more than the mutants,4 leading to an increased hyperactivity of ~2000-fold for the +2, +4, and +6 variants. Salt significantly inhibited the wild type enzyme but not the hyperactive mutants (see below). Therefore, in the presence of Mg2+, Ca2+, and 150 mM NaCl, the DNA nicking activity was progressively enhanced with each additional positive charge, cumulating in a ~6000-fold increased hyperactivity for the +6 mutant (Fig. 5, Table III). Finally, the degree of hyperactivity was greatest in the presence of 150 mM salt and the absence of Ca2+ and resulted in a >10,000-fold increase in activity over wild type for the +6 variant.

                              
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Table III
Relative nicking activity of human DNase I variants on a 32-bp DNA fragment
Experiments were carried out with 6.3 ng/ml (300 pM) of the 32P-radiolabeled 32-mer EcoRI-HindIII fragment of pBR322 under different metal ion and salt conditions. All values are normalized relative to those of wild type, which has a nicking activity of 0.22 ± 0.05 (n = 2), 0.96 ± 0.003 (n = 4), 56 ± 3 (n = 7), and 1.6 ± 0.3 (n = 2) pM DNA · min-1 · nM-1 DNase I in the presence of 1 mM Mg2+ and 150 mM NaCl; 1 mM Mg2+, 1 mM Mg2+ and 1 mM Ca2+; and 1 mM Mg2+, 1 mM Ca2+, and 150 mM NaCl, respectively. n denotes the number of separate experiments done.


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Fig. 5.   DNA nicking of the 32-bp pBR322 fragment by human DNase I variants. Radiolabeled 32-bp DNA at 6.3 ng/ml (300 pM) is incubated with DNase I in the presence of 1 mM Mg2+, 1 mM Ca2+, and 150 mM NaCl for 0.3 to 17 min. The DNase I concentration was 2700 pM for wild type (WT), 270 pM for N74K, and 27 pM for the other variants. The pattern of DNA scission shows no significant differences between the wild type and the hyperactive variants.

Salt Effects on DNA Cleavage Activity-- We further addressed the ionic effects on the DNA cleavage activity of the combination hyperactive variants because wild type human DNase I is highly sensitive to salt, and salt concentration plays a significant role in protein-DNA interactions (6, 17, 18). With a single added positive charge (N110R), the salt concentration for 50% inhibition increased from ~80 to ~200 mM NaCl in the DNA hyperchromicity assay (Fig. 6A). The double mutant E13R/N74K remained uninhibited up to 200 mM NaCl, whereas the activities of the +3 (E13R/N74K/T205K) and +4 (E13R/T14K/N74K/T205K) variants were actually enhanced by salt. The +3 variant displayed maximal activity at 150 mM NaCl, ~30% higher than in the absence of salt, whereas the +4 variant had optimal activity at 300 mM NaCl, more than twice the activity without any salt. In the absence of salt, wild type DNase I may bind DNA with optimal affinity, whereas the +3 and +4 hyperactive variants may bind to the DNA too tightly for effective catalysis. Upon addition of NaCl, the increased ionic strength should lower the substrate DNA binding affinity for DNase I, which results in significant inhibition of the native enzyme. However, in the case of the hyperactive variants, the increase in ionic strength likely permits a faster release of product DNA from the enzyme.


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Fig. 6.   NaCl effect on DNA digestion by human DNase I variants. A, the DNA hyperchromicity assay was used to follow the digestion of calf thymus DNA at the near-saturating concentration of 300 µg/ml with DNase I at increasing NaCl concentrations. The activity at a particular NaCl concentration is divided by the activity for the same variant in the absence of NaCl to obtain the percent activity. The activities in the absence of NaCl for wild type (WT), N110R, E13R/N74K, E13R/N74K/T205K, and E13R/T14K/N74K/T205K are 171 ± 10, 447 ± 31, 432 ± 36, 315 ± 10, and 300 ± 6 A260 units·min-1·mg-1 DNase I, respectively. B, EcoRI-linearized pBR322 DNA at 25 µg/ml was treated with DNase I variants in the presence of increasing NaCl concentrations and analyzed on a native agarose gel. The DNase I concentration was 270 pM for wild type (WT) and the +6 variant and 54 pM for the other two variants.

Data from the linear DNA digestion assay (Fig. 6B) are consistent with those from the hyperchromicity assay (Fig. 6A). Wild type human DNase I was sharply inhibited by salt in the linear DNA digestion assay, with ~50 and ~15% hyperchromicity activity at 60 and 150 mM NaCl, respectively. The optimal salt concentrations of the +3 and +4 variants were 150 and 300 mM NaCl, respectively. The +6 mutant also showed maximal activity at 300 mM NaCl. Therefore, the addition of one and two additional positive charges progressively alleviates the inhibitory effect of salt, whereas three to six additional basic residues require successively higher salt concentrations for optimal activity. The inhibition of DNA degradation activity by NaCl appears to be a general ionic effect because KCl and NaI also inhibited DNase I activity to a similar degree based upon data from a supercoiled plasmid digestion assay (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have systematically recombined single hyperactive mutations to generate a series of DNase I variants having from one to six additional positive charges relative to wild type. Depending on the DNA digestion conditions, variants with different numbers of basic residue substitutions possessed varying degrees of hyperactivity. In the presence of high DNA length and concentration, Ca2+, and the absence of NaCl, a modest gain in activity was found for the +1 variants (Table I, methyl green activity). Under this optimal condition for the wild type nuclease, the level of hyperactivity actually dropped with each additional basic amino acid replacement. However, under conditions that are suboptimal for wild type, which include the absence of Ca2+, increased salt concentration, and decreased DNA concentration and size, the degree of hyperactivity for the mutants relative to wild type significantly increased. Furthermore, this was progressively enhanced with successive additions of positively charged residues.

The strong conditional dependence of hyperactivity can be explained by the extremely weak affinity of wild type human DNase I for DNA. Surface plasmon resonance and gel retardation studies detected no DNA binding at up to 10 µM native DNase I concentration (data not shown). The inability of this nuclease to bind DNA tightly may explain its single-stranded nicking mechanism. Arming it with additional basic residues may prolong its duration on the negatively charged DNA and allow processive nicking of the same strand (6). The stabilization of the DNA-DNase I complex by these engineered positive charges may replace the role of the protein-stabilizing Ca2+ ion,4 yielding a greater degree of hyperactivity in the absence of this metal ion. The higher number of basic amino acids should also allow the enzyme to bind DNA tighter in the presence of NaCl. Thus, the number of additional positive charges and the salt concentration are both quite important factors because binding either too tightly or too weakly can result in inefficient DNA degradation. The association of greater hyperactivity with shorter DNA length supports the hypothesis of a processive DNA nicking mechanism utilized by the hyperactive variants (6). As the hyperactive DNase I processively digests DNA, it can slide off the ends of a smaller DNA fragment faster than a longer one. This would increase the turnover rate and yield higher nicking activity, which is determined only by the the rate of the initial nick as measured by the disappearance of the full-length substrate (Figs. 4 and 5). Finally, the correlation of increased hyperactivity with lower DNA concentration is consistent with the lower Km and Vmax for linear plasmid pBR322 nicking by the E13R/N74K mutant (Fig. 4). At lower DNA concentrations, the rate-limiting step of DNA degradation may be the formation of the enzyme-substrate complex; thus, the tighter affinity toward DNA by a hyperactive variant provides a significant advantage over wild type. On the other hand, at higher substrate levels, the dissociation of the complex could become rate-limiting, so that the stronger DNA binding by the variant would actually reduce its turnover number.

Monitoring single-stranded DNA nicking activity of the end-labeled pBR322 as a function of substrate concentration also allowed us to obtain a direct and more accurate measurement of kinetic values for DNase I than the hyperchromicity assay, which follows later stages of DNA degradation because the A260 only increases upon multiple nicking by DNase I. The absolute kinetic values cannot be reported using the hyperchromicity assay due to the nature of the polymeric substrate, the unknown number and type of different substrate binding sites, and unclear relationship between the absorbance signal and the actual catalytic events (19). On the other hand, the DNA nicking assay measures the initial rate of disappearance of linear pBR322 in a denaturing agarose gel, which is equivalent to the actual rate of catalysis because a single nick reduces the molecular weight of the substrate. Furthermore, radiolabeling permits the determination of the nicking activities at much lower DNA concentrations than the hyperchromicity assay. Accurate hyperchromicity changes due to DNase I catalysis at concentrations below 10 µg/ml were difficult to measure due to the low signal to noise ratio. However, this was not a problem using the radiolabeled DNA nicking assay, which was sensitive below 10 ng/ml DNA. Another difficulty associated with the hyperchromicity assay was that the hyperactive variants with two (E13R/N74K) or more additional basic residue substitutions yielded curves that were nonhyperbolic in the absence of NaCl, making it impossible to obtain accurate Km values.4

Electrostatic potential plays an important role in the interactions of macromolecules and is especially important for DNA-binding proteins (20); both specific local and global long-range electrostatic interactions can occur. Studies on super-repressor mutants have shown that the introduction of positively charged amino acid side chains or the removal of negatively charged ones at the protein-DNA interface increases the affinity for the operator DNA, both by increasing the association rate and by decreasing the dissociation rate (21-23). For example, single charge reversals in the lambda  and trp repressors resulted in ~600- and ~10-fold increases in affinity for operator DNA, respectively (21, 22); in the case of the trp repressor, models of electrostatic potentials that address both direct local interactions and long-range electrostatic forces have been investigated (24). In addition to providing favorable electrostatic interactions with the phosphate backbone, the introduction of a basic side chain can also significantly increase binding affinity to DNA through favorable hydrogen bonding interactions to specific bases, as seen in the structure of the engrailed Q50K homeodomain-DNA complex (25).

Our work on DNase I has shown that increasing the local electrostatic attraction toward DNA by adding specific additional positively charged residues not only improved binding affinity but also improved functional activity (6). Consistent with this observation, alanine replacement of two arginines (Arg41 and Arg111) that make critical contacts with DNA greatly reduced the DNA cleavage activity of human DNase I (7). Furthermore, the introduction of positively or negatively charged residues at sites distal to the DNA interface did not result in variants with different specific activity (5), indicating that the engineered electrostatic interactions are specific and not global. However it is interesting to contemplate the possibility of engineering favorable long-range electrostatic interactions, perhaps by decorating the appropriate surface of DNase I with either positively or negatively charged residues that might polarize the enzyme resulting in enhanced binding and/or activity (24).

The dramatic increase in activity obtained by the hyperactive human DNase I variants for degrading low molecular weight DNA at low DNA concentrations under the physiological conditions of 1 mM Mg2+, 2.5 mM Ca2+, and 150 mM NaCl (Table III) suggests that these may be of potential value for therapeutic applications. Macanovic et al. (4) recently demonstrated that treatment in a murine model of systemic lupus erythematosus with wild type murine DNase I at the relatively high dose of 7.5 mg/kg/day prolonged murine survival by 30%, as well as having beneficial effects on markers of renal function. The premise of DNase I treatment for systemic lupus erythematosus is to hydrolyze the extremely low levels of serum DNA of ~200 ng/ml5 to oligonucleotides that are small enough to prevent the formation of immune complexes or to degrade preexisting ones. Therefore, the dramatic improvement displayed both in vitro as presented here and ex vivo1 by the hyperactive variants may translate to a much more potent agent in vivo for systemic lupus erythematosus therapy. For the treatment of cystic fibrosis, only a modest enhancement over wild type might be expected because the target DNA is present at much higher concentrations of ~2 mg/ml and may only require more limited degradation of the high molecular weight DNA present to lower the viscoelasticity of cystic fibrosis sputum and improve lung function.

    ACKNOWLEDGEMENTS

We thank D. Suck for providing human DNase I coordinates; C. Eigenbrot and J. Dwyer for molecular modeling; D. Sinicropi, T. Dodge, G. Lee, M. Dwyer, R. Erickson, J. Ulmer, and L. O'Connell for technical expertise and helpful discussions; Genentech's Assay Services Group for technical assistance; M. Vasser, P. Jhurani, and P. Ng for oligonucleotide synthesis; and D. Wood for graphics.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Protein Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-1166; Fax: 650-225-3734; E-mail: lazarus.bob{at}gene.com.

1 C. Q. Pan, T. H. Dodge, D. L. Baker, W. S. Prince, D. V. Sinicropi, and R. A. Lazarus, submitted for publication.

2 The terms +1 variant, +2 variant, ... , +6 variant refer to human DNase I mutants having one, two, ... , six additional positively charged residues compared with the wild type.

3 The abbreviations used are: bp, base pair(s); TBE, tris-borate EDTA (90 mM Tris borate and 2 mM EDTA).

4 C. Q. Pan and R. A. Lazarus, unpublished results.

5 D. Sinicropi, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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