DNA synthesis in vitro : proceedings of the Second Annual Harry Steenbock Symposium held in Madison, Wisconsin on July 10-12, 1972 / edited by R.D. Wells and R.B. Inman.

  • Harry Steenbock Symposium
Date:
©1973, [1974]
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Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: DNA synthesis in vitro : proceedings of the Second Annual Harry Steenbock Symposium held in Madison, Wisconsin on July 10-12, 1972 / edited by R.D. Wells and R.B. Inman. Source: Wellcome Collection.

    81/512 (page LXXVII)
    - ^;;R,A^C-Ì\. , LINì.OLN'S !NN Jf i FI E L J i ^^YDjюLYSIS DURING DNA SYNTHESIS BY Т4 DNA POLYMERASE 49 Liti- ARY as the B22 nuclease) was shown to consist of a smgte-pefyprpTioe with a molecular weight of about 80,000 (19). In addi¬ tion, the B22 nuclease was shown to be an incomplete fragment of the wild type enzyme, by cochromatography on Dowex 50W of tryptic peptides of the two enzymes which had been previously labeled with radioactive iodoacetic acid (Fig. 2). Thus all of the [ 'C] peptides from the B22 nuclease coincided with [^H] peptides of the wild type protein, while there were several peaks containing predominantly peptides from the wild type enzyme. In this experiment 12.1 and 15.3 moles of iodoace¬ tic acid were incorporated per mole of B22 nuclease and T4 polymerase, respectively, a finding which agrees well with their half-cystine contents of 11.4 (19) and 15 (9). Under conditions where enzyme is limiting, both the wild type and the B22 nucleases remove all of the label at the 3' terminus of DNA during the initial stages of the degradation (19). This suggests that both enzymes degrade from the 3' chain end randomly rather than processively (one chain at a time) (17). The end products of the degradation in each case are 5'-mononucleotides (9) and di- and trinucleotides originating from the 5' end of the DNA chain (19). The wild type and the mutant enzyme each degrade short oligonucleo- FRACTION Fig. 2. Cochromatography of the carboxymethylated tryptic peptides from the B22 nuclease and the T4 DNA polymerase on Dowex 50-W. The B22 nuclease (143 |jLg) and the T4 DNA polymerase (95 (ig) were reduced separately by incubation in 6.4 M urea, 0.4 MTris-HCl (pH 8.3), 0.8 mM EDTA, and 5 mM dithiothreitoi, and then carboxymethy¬ lated by the addition of iodo['''C]- and iodo['H]acetic acid, respectively. The labeled proteins were combined, precipitated with cold acetone-1 N HCl, (39: 1, v/v), and digested exhaustively with trypsin. The total digest was chromatographed at 38°C on Dowex 50W-X2. The detailed method is described in reference 19. Reproduced from reference 19.