Purification and Partial Characterization of the Principal ...

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May 16, 1977 - L. BRUCE MILLS,' ERIC J. STANBRIDGE,2 W. DAVID SEDWICK,' AND DAVID KORN'*. Laboratory ofExperimental Oncology, Department ...

JOURNAL OF BACTERIOLOGY, Nov. 1977, p. 641-649 Copyright c 1977 American Society for Microbiology

Vol. 132, No. 2 Printed in U.S.A.

Purification and Partial Characterization of the Principal Deoxyribonucleic Acid Polymerase from Mycoplasmatales L. BRUCE MILLS,' ERIC J. STANBRIDGE,2 W. DAVID SEDWICK,' AND DAVID KORN'* Laboratory of Experimental Oncology, Department of Pathology, Stanford University School of Medicine, Stanford, California 94305,1 and Department of Medical Microbiology, University of California, Irvine, College of Medicine, Irvine, California 926642 Received for publication 16 May 1977

In this report we present the first description of the isolation and partial characterization of the deoxyribonucleic acid (DNA) polymerase activity from two species of Mycoplasmatales, Mycoplasma orale type 1 and M. hyorhinis. We have identified only a single DNA polymerase species in the mycoplasma crude extracts, and the enzymes from the two organisms are very similar in their structural and enzymatic properties. The purified polymerase from each source has a specific activity of >50,000 U/mg of protein, a sedimentation coefficient of 5.6s, and an estimated molecular weight by gel filtration of 130,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the most highly purified M. orale fraction contains a single major protein band of 130,000 daltons, which we believe may represent the polymerase protein. The enzymes are most reactive with gapped (activated) DNA and show a marked preference for this primer template over oligodeoxyribonucleotide-initiated homoribo- or homodeoxyribopolymers. The most purified preparations are devoid of contaminating endonuclease activity and also appear to lack associated 5' -* 3'- or 3' - 5'-exonuclease activities, as determined by highly sensitive assays. The absence of the 3' -* 5'exonuclease is particularly remarkable in that this activity is essentially ubiquitous among the DNA polymerases that have thus far been characterized from procaryotes.

The Mycoplasmatales comprise a group of microorganisms that are the smallest known free-living procaryotes. They are characterized by their minute size (0.25 to 1.0 ,um in diameter), the absence of a cell wall, and a relatively small genome (5 x 101 to 10 x 101 daltons) of presumably limited complexity that has been shown to be a single, circular, double-stranded deoxyribonucleic acid (DNA) molecule (14, 19, 24). Limited studies to date have indicated that mycoplasma DNA replication appears to be semiconservative and to proceed unidirectionally from one or a few growing-point regions (21) that may be in a membrane-associated complex (22). The capacity of at least one Acholeplasma species to carry out light and dark repair of ultraviolet-irradiated DNA has also been described (23). Apart from these few published reports, however, almost nothing is yet known about the biochemical basis of DNA replication in these organisms. Aside from their intrinsic interest, mycoplasmas have increasingly been recognized as common and troublesome contaminants of cell cultures, difficult to detect and difficult to eradicate (19, 24). Moreover, there is now abundant

evidence that such contamination, often unrecognized, can have profound effects on cultured cell properties, including alterations of metabolic properties, induction of chromosomal aberrations, and modifications of cellular morphology that can be mistaken for virus-provoked cytopathic effects (19, 24). In view of the intensive effort that is currently being expended in many laboratories on the identification and characterization of DNA polymerase activities from a variety of cultured eucaryotic cells, we felt it was important to document the properties of the DNA polymerases of representative Mycoplasmatales. In this paper, we present the purification and partial characterization of the principal DNA polymerase activity that we have isolated from crude extracts of two Mycoplasma species that are well-recognized cell culture contaminants, Mycoplasma orale type 1 and M. hyorhinis. In corroboration of the potential value of this undertaking, we note that at least one reported species of eucaryotic DNA polymerase, mitochondrial D-DNA polymerase from HeLa cells (7, 28), has recently been suggested to be a possible mycoplasma activity resulting from

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fuged at 10,000 rpm for 20 min at 4°C. The pellet, which contained organisms and particulate constituents from the growth medium, was washed twice with phosphate-buffered saline (PBS) and then stored frozen at - 70°C as an unpurified mycoplasma pellet. Initial efforts to isolate the DNA polymerase MATERIALS ANI) METHOI)S activity from such pellets revealed unpredictable Materials. Unlabeled deoxyribonucleotides were variations in column chromatographic behavior, purchased from P-L Biochemicals; 3Hldeoxythymi- which we attributed to the presence of undefined dine 5'-triphosphate (dTTP) was from New England medium components in the crude extract. We thereNuclear Corp.; calf thymus DNA was from Calbi- fore introduced an additional procedure to purify ochem; Triton X-100 was from Beckman Instru- the mycoplasma organisms. The crude pellets were ments, Inc.; Sephadex G-25 and G-200 were from suspended in 20 ml of PBS, and 5-ml portions were Pharmacia Fine Chemicals, Inc.; diethylaminoethyl overlayered on 30-ml discontinuous 30 to 60% (wt/ (DEAE)-cellulose (DE-52) and phosphocellulose (P- wt) sucrose gradients in PBS and centrifuged in an 11) resins were from Whatman; acrylamide, bisme- SW25.1 rotor at 10,000 rpm for 60 min. The visible thylene acrylamide, and N,N, N,' N'-tetramethyl- bands of mycoplasma were collected, pooled, susenediamine were from Bio-Rad Laboratories; BDH pended in PBS, and pelleted. The washing and sodium dodecyl sulfate (SDS) was from Gallard- centrifugation steps were repeated, and the final Schlesinger; bovine serum albumin and 7S immu- pellet was stored at -70'C as a purified mycoplasma noglobulin were from Sigma Chemical Co.; ovalbu- preparation. Just before the last centrifugation, the min was from Pharmacia; and ,3-galactosidase was PBS suspension was assayed for mycoplasma CFU. from Worthington Biochemical Corp. Coomassie The majority of harvests yielded a total of 1 x 101' brilliant blue was from Schwarz/Mann, and amido to 5 x 1012 CFU. Standard I)NA polvmerase assay. The standard black lOB was from Baker. LKB ampholytes were used for isoelectric focusing. Polydeoxyadenylate assay was run for 10 min at 37TC and contained the lpoly(dA)d was from Collaborative Research, and following in a total volume of 0.25 ml: tris(hydroxypolyadenylate [poly(A)] and poly(deoxyadenylate- methyl )aminomethane Tris )-hydrochloride (pH deoxythymidylate) Lpoly(dA-dT) were from Miles 8.5), 10 mM; ,6-mercaptoethanol, 2.0 mM; bovine Laboratories, Inc. Heat-denatured calf thymus serum albumin, 50 ug; MgCl2, 20 mM; KCl, 100 DNA-cellulose (Cellex-N-1; Bio-Rad) was prepared mM; activated calf thymus DNA (20), 200 gtg/ml; (1) by D. C. Eichler, Stanford University. Oligo(dT)rm deoxyadenosine 5'-triphosphate, deoxyguanosine 5'and oligo(dT)7-[3H]deoxycytidylate (dC)7) (specific triphosphate, deoxycytidine 5'-triphosphate, and activity, 14,000 cpm/pmol) were prepared (26) by T. dTTP, 100 ,kM each; liH]dTTP, 40 mCi/mmol; and S-F. Wang, Stanford University, using d(pT)4 (Col- enzyme. In addition, 0.1% Triton X-l00 was incorlaborative Research) as initiator. Terminal deoxy- porated into the reaction with the most highly nucleotidyl-transferase (13,000 U/mg), purified from purified enzyme fractions, since it appeared to calf thymus, was a gift from R. L. Ratliff, Los enhance the reproducibility of the assays. The reacAlamos Scientific Laboratory; bacteriophage T4 tions were terminated and processed as previously DNA polymerase, phosphocellulose fraction (15) described (20). One unit of DNA polymerase activity (-15,000 U/mg), was from N. G. Nossal, National is defined as the amount catalyzing the incorporaInstitutes of Health; ColEl form I [1'C]DNA (4,000 tion of 1 nmol of labeled deoxythymidine 5'-monocpm/,tg) and PM2 form I 1IHIDNA (1,600 cpm/,ug) phosphate into acid-insoluble product in 60 min at were the gift of D. A. Clayton, Stanford University; 37'C under standard reaction conditions. Specific and specifically incised, ultraviolet-irradiated E. activity is expressed as units per milligram of procoli V:'H]DNA (1.7 x 105 cpm//Ig) was provided by tein. All enzymatic properties were determined under conditions of linearity with respect to time K. H. Cook, Stanford University. Growth and harvest of mycoplasmas. M. hvor- and protein. hinis and M. orale type I were grown in a modified Glycerol gradient centrifugation. Linear 20 to Hayflick medium (9) consisting of 85 volumes of 40% (vol/vol) glycerol gradients were prepared in 50 beef heart infusion broth (Difco). 10 volumes of mM potassium phosphate (pH 7.5) and 1 mM each unheated horse serum (Irvine Scientific), 5 volumes ethylenediaminetetraacetic acid (EDTA) and /3of a 25% stock solution of freshly prepared yeast mercaptoethanol. with or without KCl at 0.5 M. extract (Standard Brands, Inc.), and glucose and Enzyme sample (0.25 ml) was layered onto the top arginine at a final concentration of 1%J. ()wt/vol) of the gradient and overlayered with mineral oil. each. Penicillin G at a final concentration of 100 U/ Sedimentation was carried out at 50,000 rpm for 24 ml was added to prevent bacterial contamination. h at 5C in an SW50.1 rotor. Fractions of 0.25 ml Flasks containing 500 ml of prewarmed growth were collected dropwise from the bottom of the tube, medium were inoculated with 5 ml of an actively and portions were tested in the standard polymerase growing culture at a density of 10'; to 10 organisms assay. Polyacrylamide gel electrophoresis. SDS-poly-. per ml and incubated 3 to 4 days at 37°C. Growth of organisms was monitored by assay of colony-form- acrylamide gels, 3 mm in diameter and containing ing units (CFU) on agar (19). Two hours before 7% acrylamide, were prepared and run according to harvest, 50 to 100 ml of prewarmed growth medium Laemmli (11), without stacking gel. Samples were was added to each culture. The cultures were centri- prepared for electrophoresis by alternate concentra-

culture contamination (la). The properties of that enzyme are very similar to those of the highly purified mycoplasma polymerases that we describe in this report.

VOL. 132, 1977

DNA POLYMERASE FROM MYCOPLASMATALES

tion against dry Sephadex G-200 beads and dialysis versus 0.05 M Tris-hydrochloride (pH 7.2), 1% SDS, 1% f3-mercaptoethanol, and 20% (vol/vol) glycerol, until the sample volume had been reduced to 5'-exonuclease function (3). Of all the DNA polymerase activities that have been described thus far, the properties of the mycoplasma enzymes most closely resemble those of the mitochondrial D-DNA polymerase from HeLa cells (7, 28), an enzyme that has recently been proposed to be a possible artifact resulting from mycoplasma contamination of the cell cultures (la). The similarities include chromatographic behavior, response to ionic strength and sulfhydryl reagents, and relative preference for DNA and synthetic homopolymer primer templates. The mitochondrial enzyme was reported to have a molecular weight of 106,000, as estimated by gel filtration (7), but, given the limitations of the methodology, we believe the difference between that size estimate and our own of 130,000 for the mycoplasma enzymes is probably not significant. Based on the data that we have presented here, together with the finding by Bolden et al. that the D-DNA polymerase-mt species is not detected in mitochondria prepared from rat liver cells or mycoplasma-free HeLa cells, it is reasonable to conclude that the mitochondrial polymerase in question was in fact the mycoplasma enzyme. ACKNOWLEDGMENTS These studies were supported by Public Health Service grant CA-14835 from the National Cancer Institute and training grant GM-01922 to L. B. M. from the National Institute of General Medical Sciences. E. J. S. is a Special Fellow of The Leukemia Society of America.

VOL. 132, 1977

DNA POLYMERASE FROM MYCOPLASMATALES LITERATURE CITED

1. Alberts, B. M., and G. Herrick. 1971. DNA-cellulose chromatography. Methods Enzymol. 21:198-217. la.Bolden, A., G. Pedrali Noy, and A. Weissbach. 1977. DNA polymerase of mitochondria is a y-polymerase. J. Biol. Chem. 252:3351-3356. 2. Bollum, F. J. 1975. Mammalian DNA polymerases. Prog. Nucleic Acid Res. Mol. Biol. 15:109-144. 3. Chang, L. M. S. 1977. DNA polymerase from bakers' yeast. J. Biol. Chem. 252:1873-1880. 4. Cook, K. H., and E. C. Friedberg. 1976. Measurement of thymine dimers in DNA by thin-layer chromatography. II. The use of one-dimensional systems. Anal. Biochem. 73:411-418. 5. DeLucia, P., and J. Cairns. 1969. Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature (London) 224:1164-1166. 6. Friedberg, E. C., and I. R. Lehman. 1974. Excision of thymine dimers by proteolytic and amber fragments of E. coli DNA polymerase I. Biochem. Biophys. Res. Commun. 58:132-139. 7. Fry, M., and A. Weissbach. 1973. A new deoxyribonucleic acid dependent deoxyribonucleic acid polymerase from HeLa cell mitochondria. Biochemistry 12:3602-3608. 8. Gefter, M. L. 1974. DNA polymerases II and III of Escherichia coli. Prog. Nucleic Acid Res. Mol. Biol. 14:101-115. 9. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep. Biol. Med. 23 (Suppl. 1):285-303. 10. Kornberg, T., and A. Kornberg. 1974. Bacterial DNA polymerases, p. 119-144. In P. D. Boyer (ed.), The enzymes, vol. 10. Academic Press Inc., New York. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Livingston, D. M., D. C. Hinkle, and C. C. Richardson. 1975. Deoxyribonucleic acid polymerase III of Escherichia coli. J. Biol. Chem. 250:461-469. 13. Low, R. L., S. A. Rashbaum, and N. R. Cozzarelli. 1976. Purification and characterization of DNA polymerase III from Bacillus subtilis. J. Biol. Chem. 251:1311-1325. 14. Maniloff, J., and H. J. Morowitz. 1972. Cell biology of the mycoplasmas. Bacteriol. Rev. 36:263-290. 15. Nossal, N. G. 1974. DNA synthesis on a doublestranded DNA template by the T4 bacteriophage DNA polymerase and the T4 gene 32 DNA unwinding protein. J. Biol. Chem. 249:5668-5676. 16. Nossal, N. G., and M. S. Hershfield. 1971. Nuclease

17.

18.

19.

20.

21. 22. 23. 24.

25.

26.

27.

28.

29.

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activity in a fragment of bacteriophage T4 DNA polymerase induced by the amber mutant am B22. J. Biol. Chem. 246:5414-5426. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. Schaffner, W., and C. Weissmann. 1973. A rapid, sensitive and specific method for the determination of protein in dilute solution. Anal. Biochem. 56:502514. Schneider, E. L., and E. J. Stanbridge. 1975. Comparison of methods for the detection of mycoplasmal contamination of cell cultures: a review. In Vitro 11:20-34. Sedwick, W. D., T. S-F. Wang, and D. Korn. 1972. Purification and properties of nuclear and cytoplasmic deoxyribonucleic acid polymerases from human KB cells. J. Biol. Chem. 247:5026-5033. Smith, D. W. 1969. DNA replication in Mycoplasma laidlawii B. Biochim. Biophys. Acta 179:408-421. Smith, D. W., and P. C. Hanawalt. 1967. Properties of the growing point region in the bacterial chromosome. Biochim. Biophys. Acta 149:519-531. Smith, D. W., and P. C. Hanawalt. 1969. Repair replication of DNA in ultraviolet irradiated Mycoplasma laidlawii B. J. Mol. Biol. 46:57-72. Stanbridge, E. J. 1971. Mycoplasmas and cell cultures. Bacteriol. Rev. 35:206-227. Vinograd, J., J. Lebowitz, R. Radloff, R. Watson, and P. Laipis. 1965. The twisted circular form of polyoma viral DNA. Proc. Natl. Acad. Sci. U.S.A. 53:1104-1111. Wang, T. S-F., W. D. Sedwick, and D. Korn. 1974. Nuclear deoxyribonucleic acid polymerase: purification and properties of the homogeneous enzyme from human KB cells. J. Biol. Chem. 249:841-850. Wang, T. S-F., W. D. Sedwick, and D. Korn. 1975. Nuclear deoxyribonucleic acid polymerase. Further observations on the structure and properties of the enzyme from human KB cells. J. Biol. Chem. 250:7040-7044. Weissbach, A., D. Baltimore, F. J. Bollum, R. Gallo, and D. Korn. 1975. Nomenclature of eukaryotic DNA polymerases. Science 190:401-402. Eur. J. Biochem. 59:1-2. Wickner, R. B., B. Ginsberg, I. Berkower, and J. Hurwitz. 1972. Deoxyribonucleic acid polymerase II of Escherichia coli. I. The purification and characterization of the enzyme. J. Biol. Chem. 247:489-497.

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