Summary
Partially purified cell-free extracts prepared from growing cells of the yeast Saccharomyces cerevisiae catalyze DNA synthesis directed by the chimeric plasmid BTYP-2 containing the whole 2 μm DNA sequence. The biochemical properties of the reaction have been examined and inactivation studies indicate that DNA synthesis is only due to protein factors. Analysis of the products of the DNA synthesis reaction demonstrates that part of the in vitro synthesized DNA is not covalently linked to the template, thus suggesting initiation of new DNA chains. The analysis of the distribution of the labelled products at early times of reaction showed a preferential synthesis on the 2 μm portion of the BTYP-2 DNA template.
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Abbreviations
- BSA:
-
bovine serum albumin
- DTT:
-
dithiotreitol
- EDTA:
-
ethylendiaminetetraacetate
- EGTA:
-
ethylenglycol-bis(β-aminoethyl-ether)N,N′-tetraacetic acid PhcMeSO2F, phenylmethylsulfonylfluoride
- SDS:
-
sodium dodecyl sulfate
References
Badaracco G, Ginelli E, Sacchi N, Sora S, Plevani P (1981) Eur J Biochem 118:247–254
Blair DG, Sharrot DJ, Clowell DB, Helinski DR (1971) Proc Natl Acad Sci USA 68:210–214
Bollum FJ (1966) In: Cantoni G, Davies D (Eds) Procedures in Nucleic Acids Research. Hayer and Row, New York, pp 296 -300
Challberg MD, Kelly TJ (1979) Proc Natl Acad Sci USA 76:655–659
Chang LMS (1977) J Biol Chem 252:1873–1880
Chang LMS, Lurie K, Plevani P (1979) Cold Spring Harbour Symp Quant Biol 43:587–595
DePamphilis ML, Wassarman PM (1980) Ann Rev Biochem 49:627–666
Fisher H, Hinkle DC (1980) J Biol Chem 255:7956–7964
Hartley JL, Donelson JE (1980) Nature 286:860–864
Hartwell LH, Mortimer RK, Culotti J, Culotti M (1973) Genetics 74:267–286
Hinkle DC, Richardson CC (1975) J Biol Chem 250:5523–5529
Hollenberg CP (1978) Mol Gen Genet 162:23–34
Jazwinski SM, Edelman GM (1979) Proc Natl Acad Sci USA 76:1223–1227
Johnston LH (1979) Mol Gen Genet 175:217–221
Johnston LH, Game JC (1978) Mol Gen Genet 161:205–214
Kojo H, Greenberg BD, Sugino A (1981) Proc Natl Acad Sci USA 78:7261–7265
Kornberg A (1980) In: DNA replication. Freeman and Company, San Francisco
Oertel W, Goulian M (1977) J Bacteriol 132:233–246
Piperno J, Alberts BM (1978) J Biol Chem 253:5174–5179
Plevani P, Chang LMS (1977) Proc Natl Acad Sci USA 74:1937–1941
Plevani P, Chang LMS (1978) Biochemistry 17:2530–2536
Plevani P, Badaracco G, Chang LMS (1980) J Biol Chem 255:4957–4963
Scott JF (1980) In: Alberts BM (ed) Mechanistic Studies of DNA Replication and Genetic Recombination, ICN-UCLA Symposia on Molecular and Cellular Biology, vol XIX, pp 379–388
Shaller H, Otto B, Nusslein V, Huf J, Hermann R, Bonhoeffer F (1972) J Mol Biol 63:183–200
Sinha NK, Morris CF, Alberts BM (1980) J Biol Chem 255:4290–4303
Wickner W, Brulag D, Schekman R, Kornberg A (1972) Proc Natl Acad Sci USA 69:965–969
Wintersberger E (1978) Eur J Biochem 84:167–172
Wyers F, Sentenac A, Fromageot P (1976) Eur J Biochem 69:377–383
Zannis-Hadjopoulos M, Persico M, Martin RG (1981) Cell 27:155–163
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Abbreviations: The abbreviations used are those recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN).
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Plevani, P., Capucci, L., Ginelli, E. et al. DNA synthesis catalyzed in vitro by yeast extracts using A 2 μm DNA containing plasmid as template for enzymatic DNA synthesis. Curr Genet 6, 47–54 (1982). https://doi.org/10.1007/BF00397641
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DOI: https://doi.org/10.1007/BF00397641