Nucleic Acids Research

Cell growth

Crithidia fasciculata Cf-C1 cells (17) were grown in a 150 l fermentor using AE80M media (18) supplemented with 100 µg/ml tryptophan, 10 µg/ml hemin and 100 µg/ml streptomycin at 28°C. The cells were harvested, washed and stored at -70°C as described previously (19).

RNase H assays

RNase H assays were carried out in 50 µl reaction volume. The substrate poly([³H] rA)·poly(dT) was synthesized as described (20) except [³H]ATP was substituted for [³²P]ATP. The reaction mixture contained 50 mM Tris-HCl, pH 8.0, 2.5 mM 2-mercapto-ethanol, 0.5 mM MnCl₂, 10% glycerol, 50 µg/ml bovine serum albumin (BSA), 40 mM (NH₄)₂SO₄ and 20-30 pmol of substrate. After incubation for 15 min at 28°C, 50 µl of 1 mg/ml calf thymus DNA and 150 µl of ice cold 10% trichloroacetic acid were added to stop the reaction. The tubes were incubated for 10 min on ice and centrifuged for 10 min at 4°C in a microcentrifuge. One hundred microliters of the supernatant were counted in Scintiverse BD scintillation fluid to determine the solubilized counts. One unit of activity is the amount of enzyme required to degrade 50% of the substrate in 15 min.

RNase H activity gels

RNase H activity gel electrophoresis and renaturation was performed essentially as described (20). The assay involves protein electrophoresis on an SDS gel embedded with a poly([³²P]rA)·poly(dT) substrate. The proteins are then renatured and RNase H activity is detected as a clearing on an autoradiogram of the gel after an acid wash. Whole cell extracts from wild-type and a RNH1 null mutant (20) were made by washing 3-4 × 10⁸ cells in phosphate-buffered saline (PBS), centrifuging, resuspending the cells in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 1% SDS, 20% glycerol and 300 mM NaCl) and centrifuging out the debris. For kinetoplast isolation from wild-type or the RNH1 null mutant C.fasciculata, cells were disrupted either by treatment with zymolyase (6) or by a Stansted Disrupter with similar results. Kinetoplasts were isolated by renographin gradient centrifugation (21). The kinetoplasts were then lysed in lysis buffer. The protein concentrations of the extracts were determined by the bicinchoninic acid method using a Pierce reagent kit according to the manufacturer's instructions.

Analysis of RNase H reaction products

RNase H reactions were prepared using poly([³²P]rA)·poly(dT) substrate. Following incubation at 28°C, EDTA was added to 10 mM to stop the reactions. The reactions were dried in a Speedvac concentrator (Savant) and resuspended in 98% formamide. Twenty percent of the reaction was applied to PEI-cellulose. Standards were 16 pmol [³²P]ATP and 240 ng AMP. The samples were separated by chromatography with 400 mM NH₄HCO₃ buffer for 2-3 h. AMP was visualized by ultraviolet illumination and the ³²P-labeled products were visualized by autoradiography.

Protein purification

All purification steps were carried out at 4°C unless otherwise specified. RNase H activity, as measured by the degradation of a poly([³H]rA)·poly(dT) substrate, was assayed during the purification. Cells (120 g) were thawed in 500 ml of H₂O with stirring; NP-40 was then added to 0.33%. The cells were stirred for an additional 2 min at which point the solution was brought to 5 mM EDTA, 20 mM spermidine, 10 mM 2-mercaptoethanol, 2 µg/ml leupeptin and 5% ammonium sulfate. Stirring was continued until all of the salt was dissolved. The lysate was cleared of cellular debris by centrifuging for 25 min at15 000 r.p.m. in a Sorvall SS34 rotor. Residual nucleic acids were removed by DEAE-Sephadex (Pharmacia) batch elution. Two hundred ml of a 50% v/v solution of DEAE-Sephadex in buffer A (200 mM ammonium sulfate, 20 mM Tris-HCl, pH 7.5, 10% glycerol, 2 mM EDTA and 2.5 mM 2-mercaptoethanol) was drained and added to the cleared lysate and allowed to stir for 1 h. The Sephadex was removed from the solution by filtering over Miracloth (fraction 1), then the Sephadex was washed with 200 ml of buffer A; the wash was added back to fraction 1. Finally, the solution was brought to 50 mM NaPO₄, pH 7.5 and a 35-80% saturated ammonium sulfate precipitation was carried out.

The ammonium sulfate pellet was dissolved in 60 ml of buffer B (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2.5 mM 2-mercaptoethanol and 20% glycerol) and dialyzed against 4 l of the same buffer overnight. The dialyzed sample was centrifuged in a SS34 rotor for 15 min at 12 000 r.p.m. to remove any insoluble precipitate and loaded onto a 50 ml single strand DNA cellulose column (USB 14.4 mg DNA/ml cellulose), equilibrated in buffer B at a flow rate of 0.64 ml/min. The RNase H activity was eluted with a 0.6 M NaCl step in buffer B. The active fractions were pooled, brought up to the same conductivity as the phenyl Sepharose loading buffer (50 mM NaPO₄, pH 7.0, 1 mM EDTA, 2.5 mM 2-mercaptoethanol, 1.2 M ammonium sulfate and 20% glycerol) by the addition of ammonium sulfate and loaded onto a 10 ml phenyl Sepharose (Pharmacia) column at a flow rate of 0.65 ml/min. The column was eluted using a step gradient of 0.8 M ammonium sulfate, 50 mM NaPO₄, pH 7.0, 1 mM EDTA, 2.5 mM [beta]-ME and 20% glycerol.

The active fractions from the phenyl Sepharose column were concentrated in an Amicon centriprep 10 concentrator to ~1 ml. This fraction was divided into 200 µl aliquots; each aliquot was run separately on a 24 ml Superdex 75 (Pharmacia) column in 50 mM NaPO₄, pH 6.5, 10% glycerol, 1 mM EDTA, 2.5 mM 2-mercaptoethanol and 50 mM NaCl, at 1 ml/min. The active fractions were pooled and immediately loaded onto a 1 ml Mono S (Pharmacia) column in buffer C (50 mM NaPO₄, pH 6.5, 20% glycerol, 1 mM EDTA and 2.5 mM 2-mercaptoethanol) at 1 ml/min. The RNase H activity was eluted with a 0-0.5 M NaCl gradient in buffer C. The purity of the active fractions was assessed by SDS-PAGE. Protein concentrations were estimated by Bradford protein assay using a Bio-Rad protein assay kit according to the manufacturer's instructions.

DNA endonuclease substrate

The standard DNA endonuclease substrate consists of three oligonucleotides. Two of the oligonucleotides are annealed to a 30 nt base oligonucleotide such that there is a nick between them and the downstream primer has a free 5[prime] single strand tail (Fig. 4). The substrate was made exactly as described previously (15). Oligonucleotides were purified (22) and 5[prime] end-labeled with T4 polynucleotide kinase and [[gamma]-³²P]ATP (3000 Ci/mmol). In some experiments, the substrate was 3[prime] end-labeled by annealing the base and 5[prime] single strand oligonucleotides and adding [[alpha]-³²P]dCTP in an exchange reaction mediated by the Klenow fragment of E.coli DNA polymerase I. The unincorporated nucleotides were removed by gel exclusion chromatography over a 0.7 ml Sephadex G-50 (Pharmacia) column. To assemble the complete substrate, the remaining oligonucleotide(s) were added to the reaction and annealed as described (15). The oligonucleotide used to block the 5[prime] single strand end is 5[prime]-GACTGCTTGACATC-3[prime]. All oligonucleotides were synthesized on an Applied Biosystems 391 DNA synthesizer.

DNA endonuclease activity assays

The DNA endonuclease activity was assayed in 15 µl reactions containing 50 mM NaPO₄, pH 6.4, 10 mM MgCl₂, 2.5 mM 2-mercaptoethanol, 100 µg/ml BSA and 10 fmol substrate. The reactions were carried out for 30 min at 30°C and then stopped by the addition of 15 µl formamide loading buffer (95% formamide, 20 mM EDTA, 0.05% xylene cyanol FF and 0.05% bromophenol blue). The reactions were heated to 65°C, separated by denaturing polyacrylamide gel electrophoresis, and visualized by autoradiography. The products were quantitated on a Molecular Dynamics PhosphorImager. The standard for size determination for the 5[prime] end-labeled substrate was a DNA sequencing ladder. The ladder was created by 5[prime] end-labeling the 16 nt upstream oligonucleotide shown in Figure 4 and annealing it to the base oligonucletide to prime a sequencing reaction using a USB DNA sequencing kit according to the manufacturer's directions. 5[prime] end-labeled oligonucleotides were used as size markers for the 3[prime] end-labeled substrate. The sequence of the oligonucleotide markers were as follows: 16mer: CACGTTGACTACCGTC; 15mer: ACACATGAATGTGCG; 14mer: GACTGCTTGACATC; 13mer: TTGAATCCCCCTC; 12mer: GGGCGGCGACCT.

Figure 1. Substrate for the endonuclease assay. The standard substrate was made by annealing the 30 nt base, 16 nt adjacent and 34 nt 5[prime] single strand overhang oligonucleotides.

RESULTS

Sub-cellular localization

Our interest in kinetoplast DNA replication mechanisms led us to purify a kinetoplast-enriched RNase H. Minicircle light strand synthesis is RNA primed and RNase H is likely to be involved in primer removal based on evidence from other eukaryotic DNA replication systems. One RNase H gene, RNH1, was isolated previously from C.fasciculata and was shown to encode two proteins of 45 and 38 kDa; however, the intracellular localization of the two proteins is unknown (20,23). To determine whether either of the RNH1 protein products are kinetoplast specific, an RNase H activity gel analysis was performed to compare activities present in whole cell extracts with those in kinetoplast extracts. Extracts were prepared from both wild-type C.fasciculata and an RNH1 null mutant (24). Characterization of the RNH1 proteins by activity gel analysis revealed other RNase H activities present in the cell. Specifically, when both alleles of the RNH1 gene are disrupted, one predominant activity of ~32 kDa remains (Fig. 1, lane 3). This activity is enriched in kinetoplasts as compared with whole cell extracts (compare lanes 3 and 4) and is present in the kinetoplast fraction of both the wild-type and mutant strains. In addition, the 45 kDa activity encoded by RNH1 is enriched in the kinetoplast fraction in comparison with the 38 kDa activity also encoded by the RNH1 gene (compare lanes 1 and 2). The apparent kinetoplast association of the 32 kDa activity led us to purify the 32 kDa RNase H activity.

Figure 2. The localization of the RNase H activities was investigated by RNase H activity gel analysis using wild-type or RNH1 deletion strains. Lane 1, 50 µg kinetoplast extract from wild-type cells; lane 2, 50 µg whole-cell extract from wild-type cells; lane 3, 50 µg kinetoplast extract from RNH1 deletion strain; lane 4, 50 µg whole-cell extract from RNH1 deletion strain.

Purification

The 32 kDa RNase H activity was purified from wild-type C.fasciculata whole cell extracts as described in the Materials and Methods. Table 1 illustrates a typical purification. While there is low recovery of activity on the single strand DNA cellulose column, it was an important step because it separated the two activities encoded by the RNH1 gene from the 32 kDa RNase H activity. The final Mono S column resulted in a 100-fold increase in specific activity and contained only one polypeptide as shown in the Coomassie-stained protein gel (Fig. 2, lane 2).

Table 1. Purification of a C.fasciculata RNase H

Fraction	Purification step	Volume (ml)	Protein (mg)	Units (10-5)	Specific activity (10-3)
I	Cleared lysate	615	7600	24	0.315
II	(NH4)2SO4 ppt	142	3200	20	0.625
III	ssDNA cellulose	67	27	4.2	16
IV	phenyl Sepharose	9.9	5	2.9	58
V	Superdex 75	6	0.822	1.7	210
VI	Mono S	2	0.007a	1.5	21 000

^aThe protein concentration was estimated by comparison of a stained band on a protein gel with a known amount determined from amino acid composition data.

Molecular mass determination

Gel-filtration analysis of the RNase H activity in comparison with several standard proteins gives a Stoke's radius of 21.5 Å (Fig. 3A). The sedimentation constant determined by analytical centrifugation is 3.7 S (Fig. 3B). The Stoke's radius and sedimentation constant were used to calculate the molecular mass of 32.4 kDa, very close to the 32 kDa size calculated from SDS-PAGE indicating that the protein is a monomer in solution. The f/f₀ value for the RNase H protein is 1.02, suggesting that it is a nearly spherical protein in solution.

Figure 3. SDS-PAGE of purified SSE1. Lane 1, protein molecular weight markers; lane 2, pooled Mono S fraction. The arrow indicates the position of SSE1. The numbers indicate the size of the molecular weight markers in kDa.

Figure 4. Determination of the molecular mass of SSE1. (A) A plot of (-logK_av)^1/2 versus Stoke's radius. The Stoke's radius of SSE1 was determined by gel filtration on a Superdex 75 column in 50 mM Tris-HCl, pH 7.5, 100 mM KCl. Standards are horse heart cytochrome c 16.4 Å, horse heart myoglobin 17.5 Å, carbonic anhydrase 20.1 Å, ovalbumin 30.5 Å and BSA 35.5 Å. The arrow indicates the elution of the RNase H activity, with a Stoke's radius of 21.5 Å. (B) A plot of s₂₀,_w (Svedberg units at 20°C adjusted for water) versus fraction number. The sedimentation coefficient was determined by sedimentation through 10-30% glycerol gradients containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mM EDTA, 2.5 mM [beta]-ME, 20 mM MgCl₂, 2 µM leupeptin and 1 mM benzaminidine. The gradients were centrifuged in a Beckman SW50.1 rotor, 42 000 r.p.m. for 19 h at 4°C. The standards are cytochrome c 2.1 S, E.coli DNA polymerase I 5.6 S and sweet potato [beta]-amylase 8.9 S. The arrow indicates RNase H activity, with a sedimentation coefficient of 3.7 s. The standards were assayed either by nick translation, SDS-PAGE, or A₄₀₅.

Endonuclease activity

In addition to the RNase H enzymes shown to be involved in primer removal, many other enzymes have RNase H activities in addition to other nucleic acid processing activities. We therefore examined the purified enzyme for other nucleic acid processing activities in addition to RNase H activity and found that the enzyme possesses structure-specific DNA endonuclease activity similar to the endonuclease activity exhibited by members of the FEN family of 5[prime] single strand flap endonuclease (14,15,25). The FEN family of enzymes cleave off a 5[prime] single strand overhang in an endonucleolytic manner (15,25). The substrate in Figure 5 was synthesized and 5[prime] end-labeled on the 5[prime] single strand oligonucleotide. The enzyme cleaved the substrate endonucleolytically releasing the 5[prime] tail. The activity requires either Mn²⁺ or Mg²⁺ and is inhibited by EDTA (Fig. 5). The cleavage product is 21 nt long indicating that the enzyme cleaves 3[prime] of the first paired nucleotide.

Figure 5. Specific endonucleolytic degradation of the substrate. The endonuclease activity of SSE1 was analyzed using the substrate drawn above the lanes and seen in Figure 1. The units are expressed in terms of the RNase H activity as explained in Table 1. -, no enzyme; +, 0.9 U of enzyme from fraction VI was added. Reactions contained 10 mM MgCl₂, 0.5 mM MnCl₂ or 1 mM EDTA. Numbers indicated size of bands in nucleotides. The 5[prime] ³²P-label is indicated by the asterisk.

Free 5[prime] end requirement

Like human FEN-1 and calf RTH-1, SSE1 endonuclease activity requires a substrate with a free 5[prime] single strand end (26,27). Annealing an oligonucleotide complementary to the 5[prime] single strand overhang, leaving 6 nt unpaired at the base of the overhang, inhibits cleavage of the substrate as shown in Figure 6. In these reactions, the 5[prime] single strand overhang oligonucleotide (Fig. 4) was 3[prime] end-labeled. Unlike the substrate with a free 5[prime] end, the blocked substrate, with a 14 base oligonucleotide annealed to the 5[prime] single strand end is not cleaved by SSE1. The free 5[prime] single strand end was shown to be required for enzyme loading for calf RTH-1. If RTH-1 was allowed to diffuse onto the substrate, the protein could be trapped on the substrate by annealing an oligonucleotide to the 5[prime] single strand end that would make the substrate double stranded (26). It is likely that SSE1 also requires the 5[prime] single strand end for enzyme loading onto the substrate.

The product in Figure 6 is 12 nt long. Based on the results of the experiment with the 5[prime] end-labeled substrate shown in Figure 5, the product expected from a single endonucleolytic cleavage would be 13 nt in length. The difference possibly reflects 5[prime]->3[prime] exonuclease activity resulting in the removal of a single nucleotide from the 5[prime] end of the paired strand. The initial product of the reaction following single strand overhang removal is a double-stranded substrate with a 1 nt gap. Removal of the 5[prime] nucleotide at the gap would create a 2 nt gap. Such structures were found to be a less effective substrate for murine FEN-1 binding (28).

Co-elution of DNA endonuclease and RNase H activities

To demonstrate that both the endonuclease and the RNase H activities reside in the same protein, the final peak off of the Mono S column, as judged by RNase H activity, was assayed for DNA endonuclease activity. Figure 7 shows that both the RNase H and the endonuclease activities elute in the same fractions with similar profiles consistent with both activities residing in the same polypeptide.

Analysis of RNase H reaction products

Since SSE1 makes an endonucleolytic cleavage of an unpaired 5[prime] DNA strand, it was of interest to determine the cleavage specificity of the RNase H activity of SSE1. In Figure 8 the reaction products at various times after incubation of a poly([³²P]rA)·poly(dT) substrate with SSE1 were separated by thin layer chromatography (TLC). The only products visible on the autoradiogram are ribonucleoside monophosphates indicating that SSE1 functions as an exonuclease on the poly([³²P]rA)·poly(dT) substrate. This result is similar to that of human FEN1 which exhibits RNase H activity as a 5[prime] exonuclease (13).

DISCUSSION

We have purified a C.fasciculata nuclease (SSE1) having both RNase H activity and structure specific DNA endonuclease activity. These dual activities suggest that SSE1 is related to the FEN1 family of eukaryotic structure-specific nucleases which have been found to be required together with the conventional RNase H for efficient removal of RNA primers in mammalian systems (10,29). The C.fasciculata enzyme appears to be kinetoplast associated based on activity gel analysis of purified kinetoplasts and may be involved in removing RNA primers during minicircle replication.

SSE1 could function in primer removal in one of two ways. If the C.fasciculata kinetoplast RNase H1 functions similarly to E.coli and calf thymus RNase H and cannot cleave the bond at the RNA-DNA junction, the SSE1 endonuclease activity might play a role in removing the remaining ribonucleotide. Advancing DNA polymerization might displace the 5[prime]-terminus creating a substrate for SSE1 cleavage of the DNA portion of the displaced strand. Alternatively, SSE1 may be capable of completely removing an RNA primer without the help of a separate RNase H enzyme since SSE1 has both RNase H and structure-specific endonuclease activities. Calf RTH1 has been shown to perform both of these activities. It is capable of removing the remaining ribonucleotide left by calf thymus RNase H (10) and it can cleave an RNA single strand flap similar to the DNA 5[prime] single strand region shown in Figure 4 (30). Human FEN1 possesses RNase H activity as a 5[prime] exonuclease; however, its activity on a 5[prime] single strand RNA flap has not been determined (13). Murine Fen1 differs from both human FEN1 and calf RTH1 as it is reported to have neither RNase H activity nor the ability to cleave an RNA 5[prime] single strand flap (15).

Like human FEN1, SSE1 appears to act as an exonuclease on the conventional poly(rA)·poly(dT) substrate. When the products of the RNase H reaction are analyzed, ribonucleoside monophosphates are the primary products, consistent with exonucleolytic cleavage of the poly(rA) strand. SSE1 is also capable of degrading the RNA strand of an RNA·M13 DNA hybrid substrate (data not shown). SSE1 may be able to function in complete primer removal since a C.fasciculata RNH1 null mutant strain is still viable although the strain has a significantly increased doubling time (24; unpublished observations).

Figure 6. Endonuclease activity requires a 5[prime] single strand end. The substrates are drawn above the reactions. -, no enzyme; +, 0.9 U of Fraction VI was added to the reactions. The numbers indicate sizes in nucleotides. The asterisks indicate the 3[prime] ³²P label.

Figure 7. The RNase H and endonuclease activities co-elute. The final RNase H activity peak from the Mono S column was assayed for endonuclease activity to show co-elution. Endonuclease activity (squares); RNase H activity (diamonds); y axis, activity in terms of percentage substrate degraded.

At any time during S phase a small fraction of the kinetoplast minicircles are undergoing replication free of the network (1,31). The recent localization of a DNA primase to the two faces of the kinetoplast disk suggests that RNA-primed initiation of minicircle replication may occur in the mitochondrial matrix at the faces of the kinetoplast disk (32). Remnants of RNA primers have been detected on newly synthesized minicircles (2,7) that have not yet been rejoined to the kDNA network suggesting that processing of these primers begins prior to reattachment of the minicircles to the network. Gaps remain in the nascent minicircles even after rejoining to the network but it is uncertain whether complete removal of RNA primers occurs prior to or following reattachment to the network. The co-localization of newly synthesized minicircles with the kinetoplast DNA topoisomerase and beta-type DNA polymerase at antipodal sites flanking the kinetoplast disk suggests that at least partial filling of gaps and rejoining of minicircles to the network takes place at these sites (33,34). Isolation and characterization of mutants of SSE1 together with precise localization of the SSE1 nuclease will be required to define more precisely the role of SSE1 in C.fasciculata.

Figure 8. TLC analysis of RNase H reaction products. The reaction products of an RNase H reaction. Using a poly([³²P]rA)[bull]poly(dT) substrate were analyzed by TLC. Lane 1, AMP; lane 2, [³²P]ATP; lane 3, undigested substrate; lanes 4-6, time course with 1.0 U of enzyme, lane 4, 5 min; lane 5, 15 min; lane 6, 30 min.

REFERENCES

1. Englund,P.T. (1979) J. Biol. Chem., 254, 4895-4900. MEDLINE Abstract

2. Birkenmeyer,L., Sugisaki,H. and Ray,D.S. (1987) J. Biol. Chem., 262, 2384-2392. MEDLINE Abstract

3. Kitchin,P.A., Klein,V.A., Fein,B.I. and Englund,P.T. (1984) J. Biol. Chem., 259, 15532-15539. MEDLINE Abstract

4. Kitchin,P.A., Klein,V.A. and Englund,P.T. (1985) J. Biol. Chem., 260, 3844-3851. MEDLINE Abstract

5. Sheline,C., Melendy,T. and Ray,D.S. (1988) Mol. Cell. Biol., 9, 169-176.

6. Birkenmeyer,L. and Ray,D.S. (1986) J. Biol. Chem., 261, 2362-2368. MEDLINE Abstract

7. Ntambi,J.M., Shapiro,T.A., Ryan,K.A. and Englund,P.T. (1986)J. Biol. Chem., 261, 11890-11895. MEDLINE Abstract

8. Perez-Morga,D. and Englund,P.T. (1993) J. Cell Biol., 123, 1069-1079. MEDLINE Abstract

9. Huang,L., Kim,Y., Turchi,J.J. and Bambara,R.A. (1994) J. Biol. Chem., 269, 25922-25927. MEDLINE Abstract

10. Turchi,J.J., Huang,L., Murante,R.S., Kim,Y. and Bambara,R.A. (1994) Proc. Natl Acad. Sci. USA, 91, 9803-9807. MEDLINE Abstract

11. Goulian,M., Richards,S.H., Heard,C.J. and Bigsby,B.M. (1990)J. Biol. Chem., 265, 18461-18471. MEDLINE Abstract

12. Ishimi,Y., Claude,A., Bullock,P. and Hurwitz,J. (1988) J. Biol. Chem., 263, 19723-19733. MEDLINE Abstract

13. Robins,P., Pappin,D.J., Wood,R.D. and Lindahl,T. (1994) J. Biol. Chem., 269, 28535-28538. MEDLINE Abstract

14. Zhu,F.X., Biswas,E.E. and Biswas,S.B. (1997) Biochemistry, 36, 5947-5954. MEDLINE Abstract

15. Harrington,J.J. and Lieber,M.R. (1994) EMBO J., 13, 1235-1246. MEDLINE Abstract

16. Siegal,G., Turchi,J., Myers,T. and Bambara,R. (1992) Proc. Natl Acad. Sci. USA, 89, 9377-9381. MEDLINE Abstract

17. Birkenmeyer,L., Sugisaki,H. and Ray,D.S. (1985) Nucleic Acids Res., 13, 7101-7118.

18. Melendy,T. and Ray,D.S. (1987) Mol. Biochem. Parasitol., 24, 215-225. MEDLINE Abstract

19. Melendy,T. and Ray,D.S. (1989) J. Biol. Chem., 264, 1870-1876. MEDLINE Abstract

20. Ray,D.S. and Hines,J.C. (1995) Nucleic Acids Res., 23, 2526-2530. MEDLINE Abstract

21. Simpson,L. and Simpson,A.G. (1978) Cell, 14, 169-178. MEDLINE Abstract

22. Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, NY.

23. Campbell,A.G. and Ray,D.S. (1993) Proc. Natl Acad. Sci. USA, 90, 9350-9354. MEDLINE Abstract

24. Ray,D.S. and Hines,J.C. (1995) Nucleic Acids Res., 23, 2526-2530. MEDLINE Abstract

25. Murante,R.S., Huang,L., Turchi,J.J. and Bambara,R.A. (1994)J. Biol. Chem., 269, 1191-1196. MEDLINE Abstract

26. Murante,R.S., Rust,L. and Bambara,R.A. (1995) J. Biol. Chem., 270, 30377-30383. MEDLINE Abstract

27. Wu,X., Li,J., Li,X., Hsieh,C.L., Burgers,P.M. and Lieber,M.R. (1994) J. Biol. Chem., 269, 28535-28538.

28. Harrington,J.J. and Lieber,M.R. (1995) J. Biol. Chem., 270, 4503-4508. MEDLINE Abstract

29. Waga,S., Bauer,G. and Stillman,B. (1994) J. Biol. Chem., 269, 10923-10934. MEDLINE Abstract

30. Murante,R.S., Rumbaugh,J.A., Barnes,C.J., Norton,J.R. and Bambara,R.A. (1996) J. Biol. Chem., 271, 25888-25897. MEDLINE Abstract

31. Pasion,S.G., Brown,G.W., Brown,L.M. and Ray,D.S. (1994) J. Cell Science, 107, 3515-3520.

32. Li,C.J. and Englund,P.T. (1997) J. Biol. Chem., 272, 20787-20792.

33. Ferguson,M., Torri,A.F., Ward,D.C. and Englund,P.T. (1992) Cell, 70, 621-629. MEDLINE Abstract

34. Melendy,T., Sheline,C. and Ray,D.S. (1988) Cell, 55, 1083-1088. MEDLINE Abstract

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*To whom correspondence should be addressed at: Molecular Biology Institute, 611 Circle Drive East, UCLA, Los Angeles, CA 90095-1570, USA.Tel: +1 310 825 4178; Fax: +1 310 206 7286; Email: danray@mbi.ucla.edu

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