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J Biol Chem, Vol. 273, Issue 51, 33991-33999, December 18, 1998


Drosophila Rrp1 Domain Structure as Defined by Limited Proteolysis and Biophysical Analyses*

Brian J. ReardonDagger §, Christian R. Lombardoparallel , and Miriam SanderDagger **

From the Dagger  Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and the  Macromolecular Interactions Facility, University of North Carolina, Chapel Hill, North Carolina 27599

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Drosophila Rrp1 is a DNA repair nuclease whose C-terminal region shares extensive homology with Escherichia coli exonuclease III, has nuclease activity, and provides resistance to oxidative and alkylating agents in repair-deficient E. coli strains. The N-terminal 421 amino acid region of Rrp1, which binds and renatures homologous single-stranded DNA, does not share homology with any known protein. Proteolysis by endoproteinase Glu-C (protease V8) reduces the Rrp1 protein to a single, cleavage-resistant peptide. The peptide (referred to as Rrp1-C274) begins with the sequence TKTTV, corresponding to cleavage between Glu-405 and Thr-406 of Rrp1. We determined that nuclease activity is intrinsic to Rrp1-C274 although altered when compared with Rrp1; 3'-exonuclease activity is reduced 210-fold, 3'-phosphodiesterase activity is reduced 6.8-fold, and no difference in apurinic/apyrimidinic endonuclease activity is observed. Rrp1 and Rrp1-C274 are both monomers with frictional coefficients of 2.2 and 1.4, respectively. Circular dichroism results indicate that Rrp1-C274 is predominantly alpha -helical, while the N-terminal 399 amino acids is predominantly random coil. These results suggest that Rrp1 may have a bipartite structural organization; a highly organized, globular C-terminal domain; and an asymmetric, protease-sensitive random coil-enriched N-terminal region. A shape model for this bipartite structure is proposed and discussed.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The stability and integrity of the genetic material of all organisms is constantly being challenged. Genetic instability results from potentially deleterious interactions of intracellular and extracellular agents with DNA and from errors introduced during DNA replication and recombination. Apurinic/apyrimidinic (AP)1 sites are the most common DNA lesions that contribute to genetic instability (1). AP sites arise through the spontaneous hydrolysis of N-glycosylic bonds, through disruption of the sugar-phosphate backbone by reactive oxygen species, or as a result of the removal of altered bases during the base excision repair process. Accumulation of AP sites can be cytotoxic, mutagenic, and carcinogenic if not repaired.

Four classes of AP endonucleases have been postulated that are distinguished by the structure of the DNA termini they produce (2). Class II AP endonucleases hydrolyze the phosphodiester bond immediately 5' to the AP site producing a 3'-hydroxyl DNA terminus, a suitable substrate for DNA repair polymerases. Additionally, class II AP endonucleases have 3'-phosphodiesterase and 3'-phosphatase activities that remove 3'-terminal groups that block polymerase function. These include some of the major lesions associated with radiation-induced DNA strand breaks. The importance of this group of enzymes for maintaining genomic integrity is confirmed by their presence in all organisms analyzed to date.

Drosophila Rrp1 (recombination repair protein 1) was the first higher eukaryotic class II AP endonuclease to be cloned (3). Rrp1 is a member of the family of Escherichia coli exonuclease III-like enzymes based on the homology to E. coli exonuclease III within its C-terminal 252 amino acids (33% sequence identity and 55% sequence similarity). This family of enzymes includes 13 homologues from bacteria to human (4-8). Unlike the other members of this family, Rrp1 has also been shown to carry out DNA strand transfer and single-stranded DNA renaturation in vitro, in addition to its DNA repair activities (3, 9, 10). This unique characteristic of Rrp1 maps to its N-terminal 421-amino acid region, a region that is not homologous to any known protein. The C-terminal region of Rrp1 complements cellular deficiency in E. coli exonuclease III and provides resistance to oxidative and alkylating agents in vivo (11). The amino acid distribution within Rrp1 is skewed; the N-terminal region is enriched with charged residues (13% lysine and 18% glutamic acid), while the C-terminal region has an average amino acid distribution. Together these observations suggest that Rrp1 may have a bipartite structural organization.

In this paper, we report the use of limited proteolysis to isolate the nuclease domain of Rrp1. Enzymatic comparison of the full-length and truncated forms of Rrp1 was carried out. This information facilitated an evaluation of Rrp1 domain structure/function and the potential influence of the N-terminal region on the nuclease activities of Rrp1. Hydrodynamic and circular dichroism measurements were also carried out on both full-length and truncated forms of Rrp1. Together, these data allow us to propose a shape model of Rrp1 domain structure.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enzymes-- Recombinant Rrp1 was purified as described previously (12). T4 polynucleotide kinase was purchased from New England Biolabs. Bleomycin was purchased from Sigma. Uracil DNA glycosylase was purchased from U.S. Biochemical Corp. Protease V8 was purchase from Boehringer Mannheim.

DNA-- Oligonucleotides were purchased from Oligos Etc. Inc. The sequences of oligonucleotides used to make the various nuclease assay substrates were as follows: TAEX-1, 5'-CGGCGGTGGCGGTTTTTTTTCCCCTTTTTT-3'; TAEX-2, 5'-GGGGGGAAAAAAGGGGAAAAAAAA-3'; BL1, 5'-CCCCAAAAAAAAAAAAGCAAAAAAAAAATTA-3'; BL2, 5'-TAATTTTTTTTTTCGTTTTTTTTTTTTGGGGGGGG-3'.

Appropriate oligonucleotides were 5'-end-labeled with 32P using T4 polynucleotide kinase in the presence of [gamma -32P]adenosine triphosphate (13). Labeled oligonucleotides were gel-purified (16% denaturing polyacrylamide gel) using an Elutrap electroelution system (Schleicher & Schuell). The recovery was calculated, and dsDNA substrates were prepared by annealing the labeled oligonucleotides with a 2.5-fold molar excess of complementary, unlabeled oligonucleotides. Annealing was carried out at 80 °C for 1 min in a buffer containing 100 mM Tris-HCl (pH 8.0) and 20 mM NaCl, followed by slow cooling to room temperature.

Nuclease Assays-- All reactions were carried out at 30 °C for 10 min in 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.05 mg/ml bovine serum albumin, 0.2 mM EDTA, 5 mM MgCl2, and 0.5 ng (4-8 nM) of labeled oligonucleotide in a final volume of 10 µl. The amount of enzyme used varied depending on the particular assay. Reactions were stopped by the addition of proteinase-K and EDTA to a final concentration of 0.05 mg/ml and 25 mM, respectively, and were incubated for 10 min at 42 °C. Prior to analytical gel electrophoresis, 5 µl of gel loading buffer (95% formamide, 20 mM EDTA (pH 8.0) 0.05% xylene cyanol FF, and 0.05% bromphenol blue) was added to each sample. The products were resolved on a 16% denaturing polyacrylamide gel. Dried gels were analyzed and quantitated using a Molecular Dynamics PhosphorImager.

dsDNA 3'-Exonuclease Substrate and Assay-- 5'-End-labeled TAEX-2 was annealed to TAEX-1 giving a dsDNA substrate with 3'-recessed ends. Reaction products were resolved on a 16% denaturing polyacrylamide gel. Calculation of fmol of nucleotide released/min/ng of enzyme has been previously detailed (14). Briefly, the relative fraction of each reaction product, as determined by PhosphorImager analysis, was multiplied by the fmol of oligonucleotide in the reaction and the difference in nucleotide length between the product and substrate. Total fmol of nucleotide released was calculated by summing over all of the product species.

3'-Phosphodiesterase Substrate and Assay-- Preparation of an oligonucleotide substrate with a 3'-terminal phosphoglycolate has been described previously (15). Briefly, BL1 contains a unique GpC dinucleotide sequence that is preferentially cleaved by bleomycin to leave a 3'-phosphoglycolate (16, 17). 5'-End-labeled BL1 was annealed to equimolar amounts of BL2, precipitated, and resuspended in 50 mM NaHPO4 (pH 7.2) and 50 mM NaCl. On ice, the sample was incubated with 0.4 mM dithiothreitol, 100 µM bleomycin, and 100 µM iron(II) ammonium sulfate for 30 min. After the addition of 0.5 volume of formamide loading buffer, the 3'-phosphoglycolate oligonucleotide, BL1-17PG, was purified as stated above. BL1-17PG was annealed to BL2. The reaction products were resolved on a 16% denaturing gel. The product BL1-17OH migrates with slower mobility than the substrate BL1-17PG. The relative specific activity was calculated by determining the amount of enzyme necessary to produce 50% product.

Proteolytic Digestion Reactions-- Protease V8 digests were carried out at 23 °C for the indicated times in a buffer with 50 mM Tris-HCl (pH 8.0), 0.5 mM dithiothreitol, 10 mM NaCl, and 10% glycerol. With the exception of preparative digests, reactions were terminated by the addition of 0.5 volumes of 2× Laemmli buffer (75 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 10% glycerol, 2% SDS, 0.1% bromphenol blue) (18). Preparative digests were stopped with 1.0 mM phenylmethylsulfonyl fluoride, 0.05 mM o-phenathroline, 0.1 mM benzamidine-HCl prior to chromatography. Proteolytic fragments were resolved on 12% SDS-PAGE gels.

Purification of His6-tagged Rrp1-- Unless otherwise indicated, all buffers used in the purification contain 1.0 mM phenylmethylsulfonyl fluoride, 0.05 mM o-phenathroline, 0.1 mM benzamidine-HCl, and a 0.5 µg/ml concentration of each of the following peptides: pepstatin A, leupeptin, chymostatin, antipain, and aprotinin. Typically, a preparation started with a 12-liter culture of induced E. coli cells (BL21(DE3), Novagen). 2× YT medium (Bacto-tryptone (16 g/liter), Bacto-yeast extract (10 g/liter), and NaCl (5 g/liter), pH 7.0) was inoculated with 0.01 volume of a fresh overnight culture and grown at 37 °C. When the cells reached mid-log phase (A600 approximately 0.5) isopropyl-beta -D-galactosidase was added to 1.0 mM with continued incubation for an additional 3 h. Cells were collected by centrifugation at 3000 rpm for 20 min in a Beckman J6-M centrifuge. Cells were washed in 500 ml of cold wash buffer (phosphate-buffered saline (5.37 mM Na2PO4, 1.76 mM KH2PO4, 137 mM NaCl, and 2.68 mM KCl), 10% glycerol, and 1.0 mM EDTA) and recentrifuged at 3000 rpm for 10 min. The cell pellet was resuspended in 150 ml of sonication buffer (50 mM Na2PO4 (pH 8.0), 100 mM NaCl, 10% glycerol, 0.1% Triton X-100, and 0.1 mM EDTA) by agitation and pipetting. Cells were frozen and thawed prior to the addition of lysozyme (Boehringer Mannheim) to a final concentration of 1 mg/ml. The mixture was gently swirled on ice for 10 min. Cells were then sonicated three times while on ice using a Branson Sonifier 450 at 50% duty cycle, output control of 4, for 30 s each cycle. Insoluble material was pelleted at 20,000 × g for 20 min in a Sorvall HB-6 rotor. Supernatant was decanted, and pellet was resuspended in 150 ml of extraction buffer (sonication buffer with 500 mM NaCl and 10 mM imidazole) by pipetting. This sample was centrifuged in a Sorvall Ti-70 rotor at 55,000 rpm for 1 h. The supernatant was removed and subjected to immobilized metal affinity chromatography. Specifically, the supernatant was mixed with 6 ml of a 50% slurry of extraction buffer and nickel-nitrilotriacetic acid resin (Ni-NTA, Qiagen) and batched for 1 h by gentle mixing. This protein-resin complex was then packed into a column for washing and elution steps. Flow-through was collected, and the column was washed with 100 ml of extraction buffer. Collected flow-through was reapplied to the 3-ml nickel-nitrilotriacetic acid column. The column was washed with 100 ml of extraction buffer, and Rrp1 was eluted with elution buffer (extraction buffer with 500 mM imidazole). Peak protein fractions (1.5-ml fractions were collected), as determined by Bradford analysis, were pooled (approximately 12 ml) and dialyzed overnight against 3 liters of buffer S (50 mM Hepes (pH 7.5), 10% glycerol, 100 mM NaCl, 0.1 mM EDTA, 0.2 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 0.01 mM o-phenathroline, 0.02 mM benzamidine-HCl, and a 0.25 µg/ml concentration of each of the following peptides: pepstatin A, leupeptin, chymostatin, antipain, and aprotinin). The dialyzed material was centrifuged at 15,000 × g for 20 min to remove precipitate material. The clarified supernatant was loaded onto an Amersham Pharmacia Biotech FPLC Mono-P HR 5/5 column, equilibrated with buffer S containing 100 mM NaCl, at a flow rate of 0.5 ml/min. The column was washed with 3 ml of buffer S containing 100 mM NaCl and eluted with a 17-ml gradient from 100 to 750 mM NaCl. Peak protein fractions were pooled and reapplied to the Mono-P column under the same conditions as described previously with the exception of a slightly shallower elution gradient (22-ml gradient). Rrp1 eluted at approximately 200 mM NaCl. The peak fraction was split; the first half was used for hydrodynamic and CD studies, while the second half was used to make sufficient quantities of Rrp1-C274 for hydrodynamic and CD studies. It should be noted that recombinant His-tagged Rrp1 has been shown to be enzymatically indistinguishable from recombinant Rrp1 lacking the His tag leader peptide.2

Purification of Rrp1-C274-- Preparative V8 digests were loaded onto an Amersham Pharmacia Biotech FPLC Mono-S HR 5/5 column, equilibrated with buffer S containing 100 mM NaCl, at a flow rate of 0.5 ml/min. The column was washed with 3 ml of buffer S containing 100 mM NaCl and eluted with a 15-ml gradient from 100 to 750 mM NaCl. Rrp1-C274 eluted at approximately 325 mM NaCl.

Protein Sequence Analysis-- Protein sequencing was performed by Dr. David Klapper (Department of Microbiology and Immunology, University of North Carolina) using a Perkin Elmer/ABI Procise 491 protein/peptide sequencer with automatic conversion of derivatized amino acids to the phenylthiocarbamoyl form and on-line HPLC analysis. This instrument is capable of sequencing materials from glass fiber filters using polybrene to immobilize the sample or sequencing from polyvinylidene difluoride type membranes using a specialized cartridge and modified sequencing programs. Current reproducible detection limits are 100-300 fmol of phenylthiocarbamoyl amino acid.

Circular Dichroism Analysis-- Measurements of CD were performed on a Jasco (Easton, MD) J-600 CD spectropolarimeter equipped with a Neslab RTE-111 temperature controller. Cells of 1-mm path length were used. A 2-s time constant with 0.1-nm bandwidth was used during acquisition over a wavelength range of 195-260 nm. Buffer conditions were 10 mM potassium phosphate (pH 8.0). Five spectra were recorded and averaged for each protein. Spectra for buffer were collected in a similar manner and subtracted. Protein concentrations for CD analysis were determined spectrophotometrically using extinction coefficients calculated from amino acid composition. Predictions of secondary structure were performed, without smoothing, against a 17-protein reference set using the program, SELCON (19). The secondary structure assignments for the reference proteins were previously calculated (20).

Analytical Ultracentrifugation Analysis-- Analytical ultracentrifugation experiments were performed on a Beckman Instruments Optima XL-A analytical ultracentrifuge equipped with absorption optics. Sedimentation velocity experiments were performed at 20 °C in two-sector charcoal-Epon-filled centerpieces. Runs were performed at 50,000 rpm in a Beckman AN60 Ti rotor. Analysis of sedimentation velocity concentration profiles was accomplished with the program SVEDBERG, kindly provided by John Philo (21). This algorithm performs nonlinear least squares fits of boundaries to approximate solutions to the Lamm equation derived by Faxen (21, 22) and yields values for the sedimentation coefficient (s) and translational diffusion coefficient (D). These values were measured at several protein concentrations and extrapolated to zero concentration to obtain the values reported in Table I. For both Rrp1 and Rrp1-C274, the single species model provided the best fit of the concentration profiles. Solution masses were calculated from the experimentally measured s and D values by the Svedberg equation as follows,
M<SUB><UP>r</UP></SUB>=RTs/D(1−&ngr;&rgr;) (Eq. 1)
where Mr is the relative molecular weight, nu  is the partial specific volume, R is the gas constant (8.3144 × 107 ergs/mol), T is temperature in Kelvin, s is time in seconds, and rho  is the density of the buffer. Measurements of solution masses for Rrp1 and Rrp1-C274 were also accomplished by sedimentation equilibrium analyses in six-sector charcoal-Epon-filled centerpieces. Rrp1 and Rrp1-C274 were sedimented at 20,000 rpm for 30 h at 4 °C at three cell loading concentrations. Equilibrium was reached as evidenced by no difference between successive scans collected over 4 h after 30 h of sedimentation. Absorbance data from the three loading concentrations were analyzed globally with the program NONLIN (23) provided by the National Analytical Ultracentrifugation Facility (Storrs, CT). A number of reaction models for nonassociating and self-associating solutes were tested. For Rrp1 and Rrp1-C274, the data were best fit to the single ideal nonassociating solute model. The final solution masses were calculated from global values of S and the reduced apparent molecular weight according to the following equation (24),
M<SUB><UP>r</UP></SUB>=(&sfgr;RT)/(1−&ngr;r)ω<SUP>2</SUP> (Eq. 2)
where sigma  is the reduced apparent molecular weight, r is the radial position in cm, and omega  is the angular velocity of the rotor, (rpm·pi )/30. Frictional ratios, f/f0, were calculated using the following equations,
f=M<SUB><UP>r</UP></SUB>(1−&ngr;&rgr;)/Ns (Eq. 3)
and
f<SUB>0</SUB>=6&pgr;&eegr;(3M<SUB><UP>r</UP></SUB>(&ngr;+&dgr;)/(4&pgr;N))<SUP>1/3</SUP> (Eq. 4)
where N is Avogadro's number (mol-1), delta  is the g of H20/g of protein, and eta  is the solution viscosity. Solution densities, rho , were measured experimentally using a Mettler DA110M density meter. Estimates of delta  (25), nu  (26), and extinction coefficients (27) were calculated using the SEDNTRP program kindly provided by Thomas Laue and John Philo (21, 28). The value of f/f0 was used to calculate the maximum axial ratio, a/b, for Rrp1 and Rrp1-C274, assuming a prolate ellipsoid shape (28, 29).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bipartite Organization of Rrp1-- It is well established that the activities of multifunctional proteins are frequently segregated into discrete structural domains of the protein. Evidence supporting the hypothesis that Drosophila Rrp1 is organized into two domains/regions is represented schematically in Fig. 1. Rrp1 derivatives truncated for each of the regions were constructed and characterized previously (12). The C-terminally truncated mutant, which was stable and soluble, is deficient in nuclease function but competent with respect to its ability to renature single-stranded DNA. Strand transfer activity of this derivative can be restored if DNA 3'-exonuclease activity is provided in trans. The biological function of the N-terminally truncated mutant was also determined previously. The C-terminal 259-amino acid region of Rrp1 is sufficient to confer resistance to oxidative and alkylating agents when expressed in a repair-deficient strain of E. coli (11). However, unlike the C-terminally truncated mutant, when produced by recombinant methods, this mutant has poor solubility properties and was not purified or enzymatically characterized.


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  		Fig. 1.   Evidence for bipartite structural organization of Rrp1. Shown is a schematic diagram of Rrp1. Selected properties of N- and C-terminal regions of Rrp1 are listed indicating regions of amino acid homology, associated activities, and amino acid distribution.

The major human AP endonuclease, HAP1, shows 27% sequence identity and 58% sequence similarity to E. coli exonuclease III (30, 31). E. coli exonuclease III is a globular protein of 2-fold alpha /beta symmetry as determined by its crystal structure (31). Based on this existing structural information and the homology alignment between HAP1 and E. coli exonuclease III, a model structure for HAP1 was constructed (30). This model displayed a remarkable degree of overall structural conservation with the structure determined for E. coli exonuclease III. Recently, this model was confirmed when the crystal structure for HAP1 was solved (31). We predicted that the C-terminal region of Rrp1, which shows 37% sequence identity and 55% sequence similarity to E. coli exonuclease III, should, like HAP1, share structural conservation with E. coli exonuclease III and exist as a discrete globular domain. Limited proteolysis has been used by others to generate polypeptide fragments that reflect independently folded domains, often providing valuable information regarding structure-function relationships (32, 33). We therefore tested whether Rrp1 would be amenable to limited proteolysis.

Fig. 2 shows the kinetics of Rrp1 digestion by protease V8 at two substrate:protease ratios. Undigested Rrp1 is shown in lane 1; the minor peptides observed are truncated products that co-purify with Rrp1. At a substrate:protease ratio of 200:1 (w/w), five discrete peptides are initially observed; over time, the intensity of the three faster migrating peptides increased (Fig. 2, lanes 2-5). When the ratio of substrate to protease is decreased 10-fold to 20:1 (w/w), the slower migrating peptides are less abundant throughout the time course, and the single fastest migrating polypeptide accumulates as the predominant species (Fig. 2, lanes 8 and 9). This single peptide is resistant to further digestion; if allowed to incubate in the presence of protease V8 an additional 14 h, the peptide is not degraded any further (data not shown). The apparent size of this peptide, based on relative mobility during SDS-PAGE, is reasonably close to the size predicted for the, C-terminal, E. coli exonuclease III homology region of Rrp1.


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  		Fig. 2.   Rrp1 includes a stable protease V8-resistant region. Shown is the digest of Rrp1 (8 µg) by protease V8 at Rrp1:protease V8 (w/w) ratios of 200:1 (lanes 2-5) and 20:1 (lanes 6-9). At the indicated time points, a volume equivalence to 2 µg of protein was aliquoted, and the reaction was terminated. Digestion products were resolved by SDS-PAGE on a 12% polyacrylamide gel. Lane 1 shows undigested Rrp1. The mobilities of marker proteins are indicated in kilodaltons.

Protease V8 is a serine protease that hydrolyzes peptide and ester bonds at the carboxylic side of glutamic acid and aspartic acid residues. The cleavage efficiency of glutamic acid as compared with aspartic acid is approximately 3000:1 (Boehringer Mannheim). 77 potential protease V8 cleavage sites (neglecting the relatively poor cleavage after aspartic acid residues) are found within Rrp1, 58 in the N-terminal region and 19 in the C-terminal region. The results in Fig. 2 suggest that the protease-resistant peptide of Rrp1 is folded such that potential cleavage sites are inaccessible to protease V8. To test this hypothesis, digests were performed in the presence of a low concentration of SDS in order to disrupt the Rrp1 structure. Protease V8 is stable and active in the presence of 0.1% SDS (Boehringer Mannheim). However, this concentration of SDS is 20-fold higher than the concentration necessary to completely inactivate Rrp1 enzymatic functions.3 Fig. 3 shows that the presence of 0.1% and 0.01% SDS (lanes 3 and 4, respectively) eliminates the appearance of the protease-resistant peptide. In contrast, complete digestion of Rrp1 by protease V8 is observed, while the protease-resistant fragment of Rrp1 is detected in a reaction lacking SDS (Fig. 3, lane 2). Thus, the appearance of the protease-resistant peptide requires the native conformation of Rrp1 and is likely to reflect the existence of a tightly structured, discrete domain. The remainder of Rrp1 appears to be relatively protease-sensitive.


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  		Fig. 3.   Protease V8 cleavage pattern reflects Rrp1 native structure. Shown is 1 µg of Rrp1 digested by 0.05 µg of protease V8 in the presence of 0.1% SDS, 0.01% SDS, or no SDS (lanes 3, 4, and 2, respectively). Undigested Rrp1 is shown in lane 1. The protease V8-resistant fragment of Rrp1 (marked by the arrow) is evident only in the absence of SDS. The mobilities of marker proteins are indicated in kilodaltons.

The Protease-resistant Peptide Is C-terminal-- Purification of the protease-resistant Rrp1 fragment was accomplished in one chromatographic step. During chromatography on Mono-S, the peptide elutes as a single peak (Fig. 4A). Correspondingly, a single peak of nuclease activity is observed (3'-phosphodiesterase) with the same elution profile as the peptide (Fig. 4B). This suggests that the peptide has intrinsic nuclease activity and is C-terminal, since nuclease activity has been previously mapped to the C-terminal 259 amino acids of Rrp1. A conserved histidine residue absolutely required for nuclease activity is located nine residues from the C terminus of Rrp1. No protease V8 cleavage sites are found between this histidine residue and the terminal residue of Rrp1, suggesting that the protease V8-resistant peptide extends to the terminal residue of Rrp1.


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  		Fig. 4.   Protease V8 fragment has intrinsic nuclease activity. Upper panel, Mono S column fractions were analyzed by SDS-PAGE on a 12% polyacrylamide gel and visualized by silver staining. Lower panel, 3'-phosphodiesterase (3'- PDE) assay of indicated Mono S column fractions. The mobilities of substrate (s) and product (p) are indicated. Mono S fractions are as numbered (L, load; F, flow-through). The mobilities of marker proteins are indicated in kilodaltons.

The five N-terminal amino acids of the peptide were determined by automated Edman degradation to be TKTTV (Fig. 5). This sequence corresponds to a cleavage between Glu-405 and Thr-406 of Rrp1, 22 residues N-terminal to the start of the Rrp1 region homologous to E. coli exonuclease III. This determination was unambiguous; no evidence of minor component peptide cleavage products with differing N termini was detectable (Fig. 5). Thus, after extensive protease V8 cleavage of Rrp1, a homogeneous, discrete protease-resistant product is obtained. In the following discussion, Rrp1-C274 will be the name used in reference to the protease V8-resistant Rrp1 fragment, indicating that its sequence includes the C-terminal 274 residues of Rrp1.


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  		Fig. 5.   N-terminal sequence of protease-resistant fragment. Five cycles of N-terminal amino acid sequencing by Edman degradation are shown. Products were analyzed by reversed phase HPLC and compared with the retention times of known amino acid standards. Amino acid identity is indicated for each of the five cycles.

Enzymatic Characterization of Rrp1 and Rrp1-C274-- Previous studies comparing the nuclease activities of E. coli exonuclease III and Rrp1 demonstrated that both enzymes are efficient, highly active AP endonucleases (15). However, the 3'-phosphodiesterase activity and dsDNA 3'-exonuclease activities associated with Rrp1 are less active than those associated with E. coli exonuclease III (15). The 3'-exonuclease activity of Rrp1 demonstrates variable efficiency according to the sequence context of the DNA substrate; Rrp1 3'-exonuclease is approximately 1 order of magnitude less active than E. coli exonuclease III with a purine-rich substrate and up to 3 orders of magnitude less active than E. coli exonuclease III with a pyrimidine-rich substrate (14). The large N-terminal region that is unique to Rrp1 may influence the characteristics of Rrp1-associated nuclease activity. To assess this possible influence, AP endonuclease, 3'-phosphodiesterase (removal of a terminal 3'-phosphoglycolate), and 3'-exonuclease activities were compared for full-length Rrp1 and Rrp1-C274. Each of the assays was carried out over a range of enzyme:substrate ratios and repeated at least three times in order to determine relative specific activity of the two nucleases. All relative specific activity measurements were normalized with respect to the difference in size of the Rrp1 and Rrp1-C274 monomers.

Previous studies have demonstrated that Rrp1 is a class II AP endonuclease with high specific activity. Rrp1-C274 also demonstrates class II AP endonuclease activity and essentially no difference in its relative specific activity as compared with Rrp1 (Rrp1:Rrp1-C274 = 0.9) (data not shown). Fig. 6A shows that the 3'-phosphodiesterase specific activity of Rrp1-C274 is 6.8-fold lower than that for Rrp1. 3'-exonuclease activity was assayed by nucleotide release from the purine-rich strand of a dsDNA oligonucleotide substrate. This substrate was chosen, since, as mentioned above, Rrp1 3'-exonuclease cleaves purine-rich sequences much more efficiently than pyrimidine-rich sequences. 3'-Exonuclease activity of Rrp1-C274 is markedly lower (210-fold) than for Rrp1 (Fig. 6B).


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  		Fig. 6.   Nuclease activities of intact Rrp1 and Rrp1-C274. A, 3'-phosphodiesterase activity assays were carried under standard conditions. A unique oligonucleotide substrate containing a 3'-phosphoglycolate was incubated with variable amounts of enzyme as indicated. The mobilities of substrate (s) and product (p) are indicated. B, 3'-exonuclease activity. The 5'-end-labeled substrate was incubated with a variable amount of enzyme as indicated for 10 min. Relative specific activities, Rrp1:Rrp1-C274, are indicated.

Hydrodynamic and CD Studies of Rrp1 and Rrp1-C274-- In order to obtain structural evidence that Rrp1 contains bipartite domain organization, structural analyses (CD, sedimentation velocity, and sedimentation equilibrium) were performed for Rrp1 and Rrp1-C274. CD measurements of Rrp1 indicated that its spectral signature resembles that of a random coiled protein (Fig. 7A). In contrast, CD spectra of the Rrp1-C274 fragment displayed the characteristic minimum at 208 nm that indicates an alpha -helical structure (Fig. 7B). Several algorithms (34-36) were initially used to deconvolute the secondary structure elements of both proteins. Although the actual percentages of secondary structure calculated varied with each algorithm, in all cases, Rrp1-C274 consistently returned greater fractions of alpha -helical and beta -sheet structural elements, while Rrp1 returned greater amounts of random coil structures. More extensive analysis was performed with the SELCON algorithm (19). For Rrp1-C274, calculated spectra returned values of 50% alpha -helix and 11% beta -sheet. SELCON was unable to accurately calculate estimates for Rrp1, presumably due to its high content of random coil, which may not be adequately represented in the secondary structure data bases used for deconvolution.


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  		Fig. 7.   CD spectrophotometry of Rrp1 and Rrp1-C274. CD spectra for Rrp1 (A) and Rrp1-C274 (B) were collected from 260 to 195 nm at 0.1-nm intervals using a time constant of 2 s and a scan rate of 20 nm/min. Five scans were collected for each protein and for 10 mM potassium phosphate, pH 8.0, buffer. The buffer was subtracted from the protein scans. The data for both proteins are normalized to molar ellipticity.

Solution mass measurements of Rrp1 and Rrp1-C274 were performed by analytical ultracentrifugation. Sedimentation velocity analyses of both proteins exhibited apparently homogeneous boundaries (Fig. 8, A and B). Multiple models were tested for the presence of single or multiple solution components. In all cases, the boundaries were best fit to a single species. Analysis of these boundaries provided values of s and D from which molecular masses were calculated using the Svedberg equation (Table I). The calculated masses from this analysis were in close agreement with Mr calculated from amino acid sequence composition. Deviations from calculated molecular weight determined by hydrodynamic measurements are attributable to asymmetric shape, hydration of Rrp1 and Rrp1-C274, or a combination of both factors. The measurement of solution mass by the technique of sedimentation equilibrium is independent of these. Sedimentation equilibrium analysis of Rrp1 and Rrp1-C274 confirmed that both proteins are monomeric. For both Rrp1 and Rrp1-C274, data from three different loading concentrations were best fit globally to the single ideal solute model. Analyses of the concentration profiles with this model resulted in residuals that were randomized and low (Fig. 9, A and B). The values of molecular masses calculated for both proteins are in good agreement with the value of the monomer calculated from amino acid composition (Table I). These measurements permitted the calculation of frictional ratios for both proteins. Rrp1 exhibited a frictional ratio value of f/f0 = 2.2 (Table I). The combined hydrodynamic data suggest that Rrp1 is an asymmetric molecule with an axial ratio of 16.2:1 based on an "ellipsoid of revolution" where the two short axes are equal, a prolate ellipsoid model. This corresponds to a maximum length of 428 Å. Rrp1-C274 had a lower f/f0 of 1.4. Using the prolate ellipsoid model, Rrp1-C274 exhibited an axial ratio of 4.4:1, corresponding to a length of 136 Å. These data indicate that Rrp1-C274 has a more compact shape than Rrp1 but is clearly asymmetric.


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  		Fig. 8.   Sedimentation velocity of Rrp1 and Rrp1-C274. Rrp1 and Rrp1-C274 were spun in a Beckman Optima XLA analytical ultracentrifuge at a speed of 50,000 rpm for 5 h. Absorbance scans were recorded every 2 min over the course of the analysis. Overlay plots of every fifth scan for Rrp1 (A) and Rrp1-C274 (B) are shown. The boundaries of these scans were analyzed using the SVEDBERG program to extract values for s and D as described under "Materials and Methods."

                              
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Table I
Physical properties of Rrp1 and Rrp1-C274


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  		Fig. 9.   Sedimentation equilibrium of Rrp1 and Rrp1-C274. Rrp1 and Rrp1-C274, each at three cell loading concentrations were spun in a Beckman Optima XLA analytical ultracentrifuge for 30 h at 20,000 rpm to achieve sedimentation equilibrium. The concentration distribution of Rrp1 (A) and Rrp1-C274 (B) as a function of radial position were analyzed. Three concentration data sets for each protein were analyzed globally using the program NONLIN to determine the solution state of each protein. The upper portion of each panel is a residual plot. Both proteins behaved as single ideal solutes (monomers) with no evidence of oligomerization. Solution molecular weights were calculated from sigma , as described under "Materials and Methods."


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Proteins are frequently organized into discrete, independently folded domains that reflect functional segregation within the protein. Previous studies and observations have suggested that Drosophila Rrp1 has a bipartite structural organization (Fig. 1). Proteolysis of native Rrp1 generates a protease-resistant fragment (Fig. 2). This fragment consist of the C-terminal 274 amino acids of Rrp1, Rrp1-C274, and has intrinsic nuclease activity (Figs. 4-6). Rrp1-C274 is a monomeric, globular polypeptide with a frictional coefficient of 1.4, while full-length Rrp1 is a monomeric and highly asymmetric protein with a frictional coefficient of 2.2. A domain organization/shape model for Rrp1 was developed from these data and is presented in Fig. 10.


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  		Fig. 10.   Shape model of Rrp1. The model represents summaries of hydrodynamic, CD, and protease digestion results. The model is represented by two overlapping ellipses generated directly from the hydrodynamic measurements. These ellipses are "prolate" and, although depicted as two-dimensional, should be viewed as three-dimensional, with the third dimension being perpendicular to the plane of the paper and equal to or shorter than the short axis.

Gel filtration studies of Rrp12 suggest that the native protein may exist as a multimer or an extremely asymmetric monomer. As calibrated against a set of globular proteins, Rrp1 would have to be, if a monomer, a globular protein with a native molecular mass of approximately 590 kDa. However, in this work (Figs. 8 and 9), Rrp1 is shown to be a monomer of 78 kDa that is extremely asymmetric, as indicated by its frictional coefficient of 2.2. Asymmetry of this magnitude is frequently associated with fibrous structural or cytoskeletal motor proteins (i.e. ankyrins, collagens, and myosins) that are usually self-associated or are composed of multiple subunits, which stabilizes their asymmetric character (37-41).

CD data (Fig. 7, A and B) show that Rrp1 is dominated by a random coil signature that is associated with extended, unstructured polypeptide regions. In contrast, the data collected on the C-terminal portion of Rrp1, Rrp1-C274, describes a domain with very distinct character from the full-length Rrp1 protein. Specifically, the frictional coefficient of Rrp1-C274 is 1.4, typical of the reported values of known globular proteins (38). The reported value for E. coli exonuclease III is 1.15 (42). CD measurements show that Rrp1-C274 has a prevalence of alpha -helical and beta -sheet folding patterns, characteristic of structured, globular polypeptide domains. The dimensions of E. coli exonuclease III and HAP1, as determined by their crystal structures, are 45 × 40 × 40 Å and 55 × 50 × 45 Å, respectively (31, 43). Since Rrp1 shares a similar degree of amino acid identity to E. coli exonuclease III and HAP1, we predict that the structure and folding pattern of the nuclease region of Rrp1 would be comparable with these two enzymes. Thus, it is likely that the difference between the frictional coefficients of E. coli exonuclease III and Rrp1-C274, which comprises the Rrp1 nuclease domain plus 22 N-terminal residues, results from the 22 amino acids appended to the E. coli exonuclease III homology region of Rrp1-C274. In fact, these 22 amino acids may be unstructured. The shape model for Rrp1 shown in Fig. 10 has a bipartite character, since the data presented strongly support such an organization for Rrp1. The C-terminal region (Rrp1-C274) has a shape that is distinctively different from the overall shape of the native molecule, and it is structured (high content of alpha -helix and beta -sheet folding patterns) and nearly globular. Furthermore, the unstructured N-terminal region of Rrp1 almost exclusively contributes to the overall high asymmetry of the whole protein. Consistent with the model, the N-terminal region of Rrp1 is sensitive to protease digestion, while the globular structure of the C-terminal domain is insensitive to protease V8 digestion.

The model shown in Fig. 10 consists of two overlapping ellipses whose dimensions were determined by hydrodynamic data (Figs. 8 and 9). The placement of these ellipses, with respect to one another, is arbitrary, and the model could be represented by a variety of placements. As discussed above, the Rrp1 C-terminal region is highly ordered. It is therefore likely to have high occupancy in a shape or space that is accurately described by these dimensions. In contrast, the lack of structure in the large N-terminal region is not consistent with the suggestion that this portion of the molecule, or the molecule as a whole, has a high occupancy in a unique shape with well defined dimensions. Thus, for the unstructured portion of the molecule, the dimensions quoted probably represent an average over occupancy in many alternate relatively unstructured forms.

Monomers of several other proteins are known that, like Rrp1, have both a relatively large unstructured domain and a terminal structured domain. The collagens, for example, have a C-terminal region, approximately 100 amino acids in length, held together by disulfide bonds that create a looped structure (39). However, only when all three chains of the collagen molecules associate to form a trimer is a distinctive globular domain at the C terminus observed. Brain ankyrin, ankyrinB (440-kDa variant), is involved in linking the spectrin/actin network to the cytoplasmic face of proteins that include cell adhesion molecules. The structural model hypothesized for ankyrinB most closely fits our model for Rrp1 and is described as a "ball and chain" (37). AnkyrinB contains an N-terminal globular domain (approximately 110-120 Å) followed by an extensive C-terminal tail (approximately 2170 Å). Rrp1 is an unusual addition to this group of previously characterized highly asymmetric proteins in that it is neither a structural nor a self-associating or multisubunit protein.

Rrp1 nuclease activity, with the exception of AP endonuclease activity, is reduced compared with E. coli exonuclease III. The influence of the N-terminal region of Rrp1 on its nuclease functions was previously unknown. It was hypothesized that an N-terminally truncated protein, such as Rrp1-C274, might become enzymatically more like E. coli exonuclease III. Comparing Rrp1 with Rrp1-C274 shows similar 3'-phosphodiesterase and AP endonuclease activities. In contrast, dsDNA 3'-exonuclease activity of Rrp1-C274 is dramatically reduced. Previously characterized Rrp1 mutants with altered nuclease activity demonstrated corollary decreases in both dsDNA 3'-exonuclease activity and 3'-phosphodiesterase activity (44). However, there is no correlation between the alteration in these activities for Rrp1-C274. Rrp1-C274 is a mutant of a single nuclease function, and the mutation causing this activity change does not lie in the nuclease active site. This result suggests that the N-terminal region of Rrp1 might contact and stabilize the conformation of the nuclease region in a manner that influences only its dsDNA 3'-exonuclease function.

It is interesting to note that other members of the E. coli exonuclease III family have N-terminal extensions and that the homology within the E. coli exonuclease III family exists in the C-terminal region of each family member (8). With the exception of the mammalian homologues, these N-terminal extensions are each unique in amino acid sequence. However, it may be possible that these N-terminal extensions are similar in their lack of well organized structure. The crystal structure of HAP1 was determined for a truncated HAP1 protein missing the 35 N-terminal amino acids (45). Crystals of the full-length enzyme diffracted poorly and showed marked mosaicity, suggesting some type of disorder.4 This suggests that the 61 N-terminal amino acids of full-length HAP1 may exist as an unstructured region that protrudes into the solvent and extends away from the folded domain structure that is homologous to E. coli exonuclease III.

With respect to a structure-function relationship of the N-terminal region of Rrp1, it is conceivable that the random coiled nature of this region contributes to its ability to bind and renature single-stranded DNA. There is a growing family of proteins that utilize extended polypeptide structures to interact with nucleic acids (46-48). The N-terminal region of Rrp1 could function in such a manner and associate with single-stranded DNA over an extended region. This might contribute to the mechanism that allows Rrp1 to facilitate the annealing of complementary DNA sequences.

    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.

§ To whom correspondence and reprint requests should be addressed: Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-4812; Fax: 919-541-7593; E-mail: reardon{at}niehs.nih.gov.

parallel Present address: The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.

** Present address: Page One Editorial Service, 403 Carolina Circle, Durham, NC 27707.

The abbreviations used are: AP, apurinic/apyrimidinic; dsDNA, double-stranded DNA; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.

2 B. J. Reardon and M. Sander, unpublished data.

3 B. J. Reardon and M. Sander, unpublished result.

4 J. Tainer, personal communication.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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