DNA synthesis in cell-free extracts of a DNA polymerase-defective mutant.

DNA SYNTHESIS IN CELL-FREE EXTRACTS OF A DNA POLYMERASE-DEFECTI VE MUTANT Thomas Kornberg and Malcolm L. Gefter Department of Biological Sciences Columbia University New York, New York 10027 Received August 4, 1970 Summary. A DNA-synthesizing activity has been isolated from an E. mmutant defective in DNA polvmerase. Like DNA polvmerase. the svstem requires the presence of all fbur’deoxynucleoside triphosphates, magnesium ion and a native DNA template for maximal activity. The activity can be distinguished from r. coli DNA polymerase on the basis of its sensitivity to high ionic strength and to p-chloromercuribenzoate. The activity is insensitive to antiserum directed against E. col i DNA polymerase. Our results do not exclude the possibility that the activity isolated is composed of, in part, an a I tered form of DNA pol ymerase. tNTRODLJCTION The isolation of a DNA polymerase-defective mutant (I) has led to several investigations into the nature of the DNA synthetic capacity of this strain. In vitro synthesis using agar-embedded cells and an in vitro membrane-associated --activity in extracts of Pol A; ccl Is have recently been reported (2,3). We wi I I describe the isolation and properties of a soluble DNA synthesizing activity from the Pol Ai mutant. MATER I ALS AND METHODS E. col i strains JG 112: W31 IO thy-, rha-, lac-, strr, Pol A;, its parent W3l IO thyPol At and JG 142: W3l 10 met E-, thy+, Pol A4 (temperature sensitive polymerase) were obtained from Dr. J. Gross. Purified g. coli DNA polymerase, an enzymatically active tryptic fragment of g. col DNA polymerase (4), and antiDNA polymerase antiserum were obtained from Dr. P. Setlow. 3 H-thymidine triphosphate (TTP) was purchased from Schwarz Bio Research and was adjusted to a specific activity of I.0 x IO5 cpm per mu mole. + For preparation of cell-free extracts from Pol or Pol A-, frozen cells (4-59) were thawed, washed twice in a solution of tris-acetate pH8.2, 0.02M; 2-mercaptoethanol, 0.005M; Mg(OACj2, O.OlM; and E.D.T.A., 0.0005M. The cells were resuspended in two volumes of the above buffer and lysed in a pressure cell (9,000 Ibs/ inchz). Debris and unbroken cells were removed by centrifugation at 12,000 XG.