©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Conserved Region in the Amino Terminus of DNA Polymerase Is Involved in Proliferating Cell Nuclear Antigen Binding (*)

(Received for publication, October 24, 1994; and in revised form, January 4, 1995)

Shan-Jian Zhang (1) Xiao-Rong Zeng (1) Peng Zhang (1) N. Lan Toomey (1) Ray-Yuan Chuang (2) Long-Sheng Chang (2) Marietta Y. W. T. Lee (1)(§)

From the  (1)Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101 and the (2)Department of Pediatrics, Children's Hospital, Ohio State University, Columbus, Ohio 43205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Synthetic peptides to selected sequences in human DNA polymerase (pol ) were used to identify the region involved in the interaction of pol to proliferating cell nuclear antigen. Peptides corresponding to sequences in five regions in the amino terminus of human pol and three in the carboxyl terminus, which are conserved with the yeast homologs of pol , were tested. These studies showed that the peptide corresponding to the N2 region (residues 129-149) selectively and specifically inhibited the PCNA stimulation of pol . This inhibition was relieved by titration with excess PCNA. The identification of the N-2 region as being involved in PCNA binding was supported by studies that demonstrated that the N2 peptide could bind PCNA. Deletion mutants of pol expressed in Sf9 cells provided evidence that the binding region for PCNA was located in the first 182 residues of the amino terminus. These studies provide reasonable evidence that residues within the region 129-149 of pol are involved in the binding site for PCNA.


INTRODUCTION

DNA polymerase (pol ) (^1)was discovered in 1976 as a new mammalian DNA polymerase that was different from the known mammalian DNA polymerases in possessing an associated 3` to 5` exonuclease activity (Byrnes et al., 1976). pol has been isolated and characterized from calf thymus and human placenta as a heterodimer of a catalytic subunit of 125 kDa and a 50-kDa subunit of unknown function (Lee et al., 1984, 1991a). The discovery of a stimulating factor for pol (Lee et al., 1984; Tan et al., 1986) eventually led to its identification as proliferating cell nuclear antigen, or PCNA (Prelich et al., 1987a, 1987b). This stimulated a remarkable advance in our understanding of the assembly of proteins required for DNA synthesis at the replication fork through the exploitation of an in vitro SV40 DNA replication assay (Li and Kelly, 1984). PCNA and pol were shown to be essential for in vitro SV40 DNA replication, and the effect of PCNA was shown to be that of a processivity factor (Weinberg and Kelly, 1989; Lee et al., 1989; Tsurimoto et al., 1990). It has been proposed that PCNA, the T4 gene 45 protein, and the beta subunit of Escherichia coli DNA polymerase III all function as sliding DNA clamps and exist as toroidal proteins that encircle the DNA strand (Kong et al., 1992; Kuriyan and O'Donnell, 1993). The assembly of the pol bulletPCNA complex at the primer terminus also requires the actions of RFC, a 5-subunit protein complex whose function is to load PCNA onto the primer-template terminus (Lee et al., 1991b).

There is a significant amount of information on the structural aspects of the DNA polymerase protein family to which pol belongs (Wang, 1991; Yang et al., 1992). Multiple sequence alignments have shown the presence of a core region that contains six highly conserved regions. It is generally accepted that a number of amino acid residues located in the core region of DNA polymerase are involved in the catalytic function of the proteins (Blasco et al., 1992, 1993; Dong et al., 1993). There is some conservation in the carboxyl-terminal regions, which contain two zinc-finger motifs (Wang et al., 1991), which are thought to be involved in DNA binding. The sites involved in the 3` to 5` exonuclease activity of DNA polymerase have also been identified (Simon et al., 1991; Morrison et al., 1991). The amino-terminal regions of the pol alpha family, however, were found to be highly dissimilar (Wang, 1991). We have cloned the cDNA for human pol and have performed multiple sequence alignments that established that human pol is closely related to yeast pol and to members of the herpesvirus DNA polymerases and is a member of the pol alpha protein family (Yang et al., 1992). These alignments allowed us to show for the first time that while it had been thought that the amino-terminal regions of the polymerases were very different, within members of this group there are some significant relationships. We had identified five regions of conservation which we have labeled N1-N5 (Yang et al., 1992; Hao et al., 1992). Our interests are directed toward structure-function aspects that are unique to pol , viz. the identification of the protein-protein interaction sites.

In this study we report the use of synthetic peptides to the sequences in the amino-terminal region to probe for the pol region that is involved in its binding to PCNA. Additional experimental approaches using deletion mutants of pol also support this hypothesis.


EXPERIMENTAL PROCEDURES

Materials

Poly(dA)bulletoligo(dT) was purchased from Midland Certified Reagent Company, Midland, Texas. [methyl-^3H]dTTP (55 Ci/mmol) for pol activity assay was from ICN Radiochemicals. Anti-PCNA monoclonal antibody was obtained from American Biotech Inc., Plantation, FL. DNA pol was purified from human placenta as described previously (Lee et al., 1991a); pol had a specific activity of 26,000 units/mg when assayed using poly(dA)bulletoligo(dT) as the template-primer. Purified PCNA stimulated pol activity about 30-fold. PCNA was the recombinant human protein, expressed in E. coli and purified to homogeneity. (^2)

Synthetic Peptides

Synthetic peptides were prepared by the Protein Chemistry Core Laboratories of Miami and University of Florida. The preparations used were pure based on the elution of a single peak at 214 nm from a C(18) reverse-phase HPLC column, and by amino acid composition. The following peptides to the conserved regions N1-N5 of human pol (Yang et al., 1992) were synthesized: N1 (84-101), N2 (129-149), N2a (129-138), N2b (139-149), N3 (244-262), N4 (276-295), and N5 (312-331). In addition, three peptides covering the last 61 residues of the carboxyl terminus were synthesized: C1 (1047-1068), C2(1069-1090), and C3(1091-1107). The peptides were dissolved in 50 mM Tris-HCl, pH 8.5, before use.

Dot Blot Analysis for the Interaction between pol Synthetic Peptides and PCNA

About 20 µg of each pol synthetic peptide was dotted onto a nitrocellulose membrane and air dried. The membrane was blocked with nonfat dry milk (5%) for 30 min and then incubated with purified PCNA (20 µg/ml) at 4 °C overnight. After washing with 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl 3 times, the membrane was incubated with PCNA monoclonal antibody at 4 °C for 6 h. The membranes were then visualized by following the same procedures as for Western blot analysis using an anti-PCNA monoclonal antibody (Zeng et al., 1994).

Assay for pol Activity

pol was assayed as described previously Lee et al. (1991a) using poly(dA)bulletoligo(dT) in a ratio of 20:1 as the template-primer and [^3H]dTTP as the nucleotide donor. The standard reaction (100 µl) contained 0.25 OD units/ml poly(dA)bulletoligo(dT) (20:1), 200 µg/ml bovine serum albumin, 5% glycerol, 10 mM MgCl(2), 25 mM HEPES, pH 6.0, 10 µM [^3H]TTP (100 cpm/pmol), and 0.2-0.4 units of pol . PCNA, where added, was 0.2 µg/assay. Incorporation of [^3H]dTTP was determined by binding of the DNA onto DE81 paper as described by Lee et al. (1991a).

Preparation of Immobilized Peptides

The N2 and N4 peptides were individually coupled to CH-Sepharose (Pharmacia Biotech Inc.). The peptides were coupled with CH-Sepharose according to the manufacturer's protocol. The same procedure was used for coupling of bovine serum albumin, which was used as a control. The activated CH-Sepharose (0.25 g) was suspended in cold 1 mM HCl for 15 min and then separated by centrifugation and washed with ice-cold 0.1 M NaHCO(3), 0.5 M NaCl, pH 8.3. The gel was then mixed with the peptide solution (2.5 mg in 1 ml of 0.1 M NaHCO(3), 0.5 M NaCl, pH 8.3), and the suspension was rotated end over end overnight. The gel was blocked by incubation with 1 ml of 0.2 M glycine, pH 8, at 4 °C for 18 h. The gel was washed with 25 ml of 0.1 M NaHCO(3), 0.5 M NaCl, pH 8.3.

Adsorption of PCNA on N2-CH-Sepharose

E. coli lysate expressing recombinant human PCNA (0.8 ml) was mixed with 40 µl of N2 or N4 coupled to CH-Sepharose, and the suspension was mixed at 37 °C for 1 h. The beads were then centrifuged down and washed 6 times with 0.1 M NaHCO(3), 0.6 M NaCl, pH 8.3. After the final wash, the beads were resuspended in 80 µl of SDS-PAGE sample buffer and boiled for 2 min (Lesley and Burgess, 1989). The supernatant was then taken directly for SDS-PAGE and Western blotting analysis using anti-PCNA antibody (Zeng et al., 1994).

Construction and Expression of PCNA, pol and pol Deletion Mutants in the Baculovirus Expression System

The cDNA for pol was that reported by Yang et al.(1992). The coding sequences for PCNA, pol , and pol mutants were inserted into the pVL1392 vector (Invitrogen Corp.). Four deletion mutants, Delta2-249, Delta186-321, Delta336-715, and Delta778-1047 and mutant Delta674-1107, in which the coding sequence for residues 674-1107 has been partially deleted or frameshifted, were constructed. (^3)Expression of the recombinant proteins was performed by infection of Sf9 cells (Ausubel et al., 1994).

Processivity Assay

The effect of synthetic peptides on the processivity of pol in the presence of PCNA was analyzed by polyacrylamide gel electrophoresis of the reaction products as described by Prelich et al. (1987a). The reaction mixtures contained 100 µg/ml of poly(dA)bulletoligo(dT) in a ratio of 40:1. The oligo(dT) was end-labeled with [-P]ATP and T4 DNA polynucleotide kinase. The total reaction volume was 60 µl. Reactions were started by the addition of 1 unit of DNA pol , 0.06 µM PCNA, and 50 µM of the individual synthetic peptides. Reactions were allowed to proceed at 37 °C for 30 min and terminated by the addition of EDTA to a final concentration of 3 mM. Salmon testes DNA was added to a final concentration of 15 µg/ml, and the samples were precipitated with ethanol. The pellets were resuspended in deionized formamide containing 1 mM EDTA, 0.1% xylene cyanole, and 0.1% bromphenol blue. These were run on 8% acrylamide, 7 M urea gels with 80 mM Tris-HCl, 80 mM boric acid, 4 mM EDTA, pH 8.3.


RESULTS

Inhibition of PCNA-stimulated pol Activity by Synthetic Peptides

The rationale for testing peptides to the amino terminus of pol as potential sites of interaction with PCNA came from observations that the pol sequence could be subdivided into three regions: an amino-terminal region (1-366) that has little relation to the amino termini of the other polymerases, a core region (367-837) that is highly conserved, and a carboxyl-terminal region (7) that possess some relationship with pol alpha (Yang et al., 1992). However, between yeast and human pol , and to a lesser extent that of the herpes simplex virus type I DNA polymerase, there were five regions that exhibited some degree of conservation in the amino terminus (Yang et al., 1992). The possibility that the conserved regions shared between the pol homologs might be involved in PCNA binding would also be consistent with the observation of heterologous interactions between mammalian and yeast PCNA and pol (Bauer and Burgers, 1988).

The peptides that were synthesized are shown in Table 1. In addition to the amino-terminal peptides, three peptides covering the most carboxyl-terminal 61 residues of pol were also synthesized. These peptides were then tested for their ability to inhibit the PCNA stimulation of pol activity using sparsely primed poly (dA)bulletoligo(dT) as a template-primer. The results are shown in Fig. 1. Of the peptides tested, only the peptide N2 exhibited an ability to inhibit the PCNA stimulation of pol . The N2 peptide inhibited 80% of the PCNA-stimulated DNA polymerase activity at a concentration of 20 µM. At a concentration of 50 µM, >95% of pol enzymatic activity was inhibited by the N2 peptide. The amino-terminal region peptides N1, N3, N4, and N5 did not inhibit the PCNA stimulation of pol (Fig. 1). The N2a and N2b peptides, which are the two halves of the N2 peptide, also did not inhibit PCNA stimulation of pol activity either singly or in combination (not shown). The carboxyl-terminal synthetic peptides did not have any significant effect on PCNA stimulation of pol , although peptide C2 exhibited a weak inhibition (35%) at a concentration of 100 µM (not shown). These results indicated that the inhibition of pol activity was due to interference with PCNA binding and was a specific rather than a nonspecific effect. Analysis of the reaction products of the pol reaction by polyacrylamide gel electrophoresis was performed (Fig. 2). These confirmed that only the N2 peptide inhibited the reaction, as shown by the loss of processivity in its presence.




Figure 1: Effects of synthetic peptides on the PCNA-stimulation of pol activity. Purified human placental pol was assayed using poly(dA)bulletoligo(dT) as the template-primer in the presence of PCNA (see ``Experimental Procedures'') and in the presence of increasing concentrations of the peptides N1, N2, N3, N4, and N5 (Table 1). Data are shown as percent of control activities in the absence of the peptide.




Figure 2: Effect of synthetic peptides on the pol processivity. The effects of peptides on the processivity of the pol reaction were examined by polyacryalmide gel electrophoresis of the reaction products as described under ``Experimental Procedures.'' The lanes show the results in terms of additions to the basic reaction mixture, which contains the template and labeled nucleotides as follows: lane1, no addition; lane2, + PCNA; lane3, + pol ; lane4, + pol + PCNA; lane5, + pol + PCNA + N1; lane6, + pol + PCNA + N2a; lane7, + pol + PCNA + N2b; lane8, + pol + PCNA + N2; lane9, + pol + PCNA + N3; lane10, + pol + PCNA + N4; lane11, + pol + PCNA + N5; lane12, + pol + PCNA + C1; lane13, + pol + PCNA + C2; lane14, + pol + PCNA + C3; lane15, 1-kilobase ladder (Life Technologies, Inc.). Peptides, where added, were at concentrations of 50 µM



Experiments to determine if the inhibition by peptide could be competed against by titration with PCNA were performed (Fig. 3). Addition of increasing amounts of PCNA reversed the inhibition. In the presence of 25 µM of peptide, 100% of pol activity was recovered by as little as 1 µM PCNA. These results showed that PCNA readily competes against the inhibitory effect of peptide N2 on PCNA-stimulated pol activity. These results support the view that N2 peptide competes with pol for binding to PCNA.


Figure 3: Reversibility of the N2 peptide inhibition of pol by saturation with PCNA. Human pol was assayed in the presence of the indicated concentrations of N2, with increasing amounts of PCNA. The data were plotted as percent of the control PCNA-stimulated activity in the absence of added peptide N2.



Demonstration of a Physical Interaction between N2 and PCNA

In order to demonstrate that the peptide N2 was capable of a physical interaction with PCNA, dot blot experiments were performed in which samples of the peptides were placed on nitrocellulose membranes, incubated with PCNA, and then subjected to the same procedure as used for Western blotting using an anti-PCNA antibody. The results show that only N2 of the peptides tested showed a positive signal (Fig. 4).


Figure 4: Dot blot analysis of the binding of PCNA to synthetic peptides. The synthetic peptides were dotted onto nitrocellulose membranes, which were then blocked and incubated with PCNA. The binding of PCNA to the membranes was then visualized by immunoblotting methods using an antibody to PCNA (see ``Experimental Procedures''). B refers to bovine serum albumin; P refers to PCNA.



The peptide N2 was immobilized on CH-Sepharose, and the support was tested for its ability to bind PCNA. An E. coli lysate expressing recombinant human PCNA^2 was treated with Sepharose beads to which either N2 or N4 had been attached, and the beads were washed, extracted with SDS buffer, and then Western blotted (see ``Experimental Procedures''). The results showed that the N2-beads were capable of binding PCNA as shown by SDS-PAGE and silver staining (Fig. 5, leftpanel) or by Western blotting (Fig. 5, rightpanel). The binding was prevented by the addition of free N2 peptide. The other peptides (Table 1) were also coupled to CH-Sepharose, but all of these except N2 gave negative results as shown for N4-CH-Sepharose in Fig. 5. Similar tests of bovine serum albumin coupled to Sepharose were also negative. In other experiments, similar results were obtained using a crude calf thymus extract in that only the immobilized N2 peptide was able to bind PCNA.


Figure 5: Binding of PCNA to immobilized N2 peptide. N2 peptide was covalently coupled to CH-Sepharose. The Sepharose beads (40 µl) were then used to bind PCNA from 0.8 ml of an E. coli lysate expressing the recombinant protein. After stringent washing to remove nonspecifically bound material, the beads were extracted with SDS-buffer and subjected to SDS-PAGE and Western blotting using an antibody to PCNA (see ``Experimental Procedures''). Leftpanel, silver staining of SDS-PAGE. Lane1, material bound to N4-CH-Sepharose; lane2, material bound to N2-CH-Sepharose; lane3, material bound to N2-CH-Sepharose from a mixture of the lysate with N2 peptide (1 mg); S, protein standards (alpha(2)-macroglobulin, 180 kDa; beta-galactosidase, 116 kDa; fructose 6-phosphate kinase, 84 kDa; pyruvate kinase, 58 kDa; fumarase, 48 kDa; lactate dehydrogenase, 36 kDa; triosephosphate isomerase, 26 kDa). Rightpanel, as in leftpanel but Western blotted with anti-PCNA antibodies. (Authentic PCNA migrates between the 26- and 36-kDa standards.)



Localization of the PCNA Binding Region of pol by Analysis of Deletion Mutations

We have recently expressed the catalytic subunit of human pol in Sf9 cells using a baculovirus vector and have also constructed a series of pol deletion mutants.^3 These were ones in which residues 2-249, 186-321, 336-715, 778-1047, and 675-1107 were deleted (see ``Experimental Procedures''). These are shown diagrammatically in Fig. 6. Sf9 insect cells were coinfected with the individual recombinant viruses expressing the deletion mutants or the full-length pol together with a baculovirus expressing human PCNA. The cell lysates were then immunoprecipitated with a monoclonal antibody against PCNA. The immunoprecipitates were then Western blotted with antibodies against pol . The results (Fig. 7) shows that the full-length pol as well as the deletion mutants were all co-precipitated with PCNA, with the exception of deletion mutant Delta2-249. Since the deletion mutant 186-321 also coprecipitated with PCNA, the results indicate the PCNA binding region must fall within residues 1-185, consistent with the location of the N2 region (residues 129-149).


Figure 6: Deletion mutants of pol . The upperbar shows a diagram of the pol sequence. The boxes represent regions that are conserved with other DNA polymerases as defined by Yang et al.(1992). Lowerbars show diagrammatically the deletions that were made.




Figure 7: Analysis of the ability of deletion mutants of pol to bind to PCNA. Sf9 cells were co-infected with recombinant baculovirus vectors for the full-length human pol sequence (7) and the deletion mutants Delta2-249, Delta186-321, Delta336-715, Delta778-1047, and Delta674-1107 together with a baculovirus vector for human PCNA. Cell lysates from each of the cultures were then immunoprecipitated with an antibody to human PCNA. The immunoprecipitates were then Western blotted with a mixture of two monoclonal antibodies to the amino- and carboxyl-terminal regions of pol . Control experiments (not shown) indicated that levels of expression of the deletion mutants were similar as determined by Western blotting of the Sf9 cell lysates. sfiDelta refers to the Delta674-1107 mutant.



These mutants have not been fully characterized, but our preliminary studies show that the possibility of interference by contaminating endogenous baculovirus or insect cell DNA polymerases can be ruled out. The full-length pol expressed in baculovirus is active and is stimulated by PCNA, while the Delta2-249 deletion mutant is not stimulated by PCNA.^3


DISCUSSION

Synthetic peptides were used to identify a site on pol that is involved in its protein-protein interaction with PCNA. Our results show that a 21-residue peptide containing the sequence of a conserved region (N2) in the amino terminus of pol is capable of inhibiting the interaction of pol with PCNA, presumably by competitive binding to PCNA. The identification of this region as the PCNA binding site is supported by the fact that this inhibition can be competed out by further addition of PCNA and by the fact that the half peptides of N2 (N2a, N2b) do not inhibit. Peptides have been usefully employed for the study of protein interaction sites, e.g. for epitope mapping and for structure-activity studies of peptide hormones (Hruby et al., 1992). The use of synthetic peptides for studies of protein-protein interaction sites in proteins has been more limited, but have been successfully used in studies of the autoinhibitory domains of the calcium/calmodulin protein kinases (Soderling, 1990).

The peptide inhibition studies are supported by the direct demonstration that the N2 peptide has the ability to bind to PCNA, either by dot blotting or by binding of PCNA to immobilized N2 peptide. Analysis of deletion mutants of pol coexpressed with PCNA in Sf9 cells by recombinant baculoviruses also confirm that the PCNA binding region is located within the first 185 residues of the amino terminus of pol . The latter result is consistent with the observations that the deletion of the first 220 amino acids of yeast pol (Brown and Campbell, 1993) result in loss of PCNA activation. The successful use of the 21-residue N2 synthetic polypeptide also indicates that the PCNA interaction site on pol is limited to this linear sequence of peptide, i.e. it does not require the full tertiary structure of the protein, and is located within a contiguous peptide sequence.

We have overexpressed human PCNA in E. coli and have shown that free PCNA at low concentration exists in a dimer-trimer equilibrium and that discrete dimer and trimer forms can be observed on gel permeation HPLC.^2 These data are consistent with the proposal that PCNA is a trimeric species that forms a toroid by analogy with the E. coli DNA polymerase III beta subunit and the T4 gene 45 protein based on sequence alignment of the three proteins (Kong et al., 1992). In the beta subunit, each of the monomers possesses three structurally similar domains. If PCNA is a trimeric species, then clearly the symmetry of the molecule dictates that there are three potential binding sites for pol , so that the possibility that PCNAbulletpol complexes involving more than one pol molecule exists.

The interaction of PCNA with the peptides (and hence pol ) is independent of the presence of DNA. We have shown that immobilized PCNA is able to bind pol in the absence of DNA. (^4)Our studies show that the binding of pol to PCNA is mediated by a region of peptide sequence within the 21-residue peptide in the amino terminus of pol region. The N2 region can be characterized by the consensus GX(4)GX(8)GX(3)YFY between human, Saccharomyces cerevisiae, and Schizosaccharomyces pombe pol . There are a number of glycine-rich motifs known, including the nucleotide binding phosphate or p-loop motif that is present in several protein families (Saraste et al., 1990) and the GXGXXG motif for the binding of adenine nucleotides of the serine protein kinases (Knighton et al., 1991). However, since the crystal structure of pol is unknown, the significance of this repeat in the N2 region is uncertain. The question is raised as to whether this site is the only site responsible for the binding of PCNA. This cannot be fully ascertained, but the strong inhibition of binding by the peptide suggests that this may indeed be the case.

These studies provide the first identification of a protein-protein interaction site in human pol in the form of its interaction site with PCNA. Given that the assembly of pol at the primer-template terminus requires a number of accessory proteins, the location and nature of these protein-interaction sites are therefore of some interest. It is now clear that the formation of the replication complex, and possibly the regulation of polymerase in the cell cycle, may involve protein-protein interactions with multiple protein partners, including replication factor C and the cyclin-dependent kinase systems. It is therefore of interest that recent studies have shown that PCNA can interact with several cyclins, cyclin-dependent kinases, and p21 (Xiong et al., 1992, 1993; Brott et al., 1993). Recently, p21 has been shown to inhibit SV40 DNA replication in vitro (Waga et al., 1994; Flores-Rozas et al., 1994) in addition to its ability to inhibit cyclin-dependent kinase. Because pol activity requires the presence of PCNA, modulation of its interaction with PCNA may be an important mechanism for the regulation of pol activity and hence of DNA replication, and a knowledge of the structural basis for these protein-protein interactions may be of significance. Further examination of the role of the N2 region in mediating the interaction of pol with PCNA is being performed by site-directed mutagenesis.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM31973 (to M. Y. W. T. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: P. O. Box 016960, University of Miami School of Medicine, Miami, FL 33101. Tel.: 305-547-6338; Fax: 305-547-3955.

(^1)
The abbreviations used are: pol , DNA polymerase ; PCNA, proliferating cell nuclear antigen; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

(^2)
P. Zhang, S. J. Zhang, Z. Zhang, J. F. Woessner, and M. Y. W. T. Lee, submitted for publication.

(^3)
S. M. Wu, P. Zhang, R. Y. Chuang, L. S. Chang, and M. Y. W. T. Lee, manuscript in preparation.

(^4)
S.-J. Zhang, X.-R. Zeng, P. Zhang, N. L. Toomey, R. Y. Chuang, L. S. Chang, and M. Y. W. T. Lee, unpublished observations.


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