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Mechanism of Synthesis of Deoxyribonucleic Acid in Vivo

THE HETEROGENEITY OF INCORPORATION OF ³²P INTO THE DEOXYRIBONUCLEOTIDYL UNITS IN ESCHERICHIA COLI

From the ¹ From the Department of Biochemistry, the Department of Urology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, and the Department of Medical Microbiology and Immunology, the Department of Biological Chemistry, and the Molecular Biology Institute, University of California, Los Angeles, California 90024

Mechanisms of synthesis of deoxyribonucleic acid in a thymine-requiring mutant strain (I) of Escherichia coli and its wild parent strain 15 were investigated by analysis of the incorporation of labeled phosphate (³²P) into free nucleotides and nucleotidyl units of DNA and ribonucleic acid. After short incubations of strain I with ³²P, the relative specific activity of the 5'-nucleotides of the DNA was: deoxythymidylate much greater than deoxyadenylate, which was approximately equal to deoxyguanylate, which was greater than deoxycytidylate. 3'-Deoxyribonucleotides from the same DNA showed only very slight heterogeneity. When 5-bromouracil and ³²P were incorporated simultaneously into DNA of strain I, they appeared together in new long segments. A high 5'-phosphate to 3'-phosphate-labeling ratio found in natural DNA only for the case of deoxythymidine 3',5'-diphosphate units existed also in the bromodeoxyuridine 3',5'-diphosphate. The findings indicate that (a) the ³²P-labeling heterogeneity among 5'-deoxyribonucleotides of E. coli DNA is determined exclusively by differences of specific activity values of proximal precursors of the DNA and (b) the precursors in vivo are activated 5'-deoxyribonucleotides, presumably the deoxyribonucleoside 5'-triphosphates. The pre-eminent labeling of 5'-deoxythymidylate or 5'-bromodeoxyuridylate of the DNA of strain I is attributable to acquisition of its phosphate directly from the rapidly renewed -phosphate of ATP, without isotope dilution inherent in passing through a large and more slowly renewed -phosphate pool and 5'-ribonucleotides. An intermediate labeling of 5'-deoxythymidylate in the DNA of the wild strain 15, with no substantial change in the heterogeneity among the other 5'-deoxyribonucleotidyl units as compared with strain I, indicates that, in strain 15, ³²P passes through the -phosphate of diluting pools of 5'-uridylate to reach the 5'-deoxythymidylate even when this strain is grown in a medium with thymine.

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