Abstract
We have directly tested the hypothesis that elevated levels of dUTP cause the formation of small DNA fragments at the replication fork of Escherichia coli. Addition of increasing levels of dUTP to lysates on cellophane discs results in an increasing number of strand scissions in the newly replicated DNA. Lysates of strains defective in dUTPase produce many more scissions at the same level of dUTP. The size distribution of Okazaki pieces obtained in vivo can be reconstituted in vitro on cellophane discs if appropriate levels of dUTP are present. Although uracil excision leads to the apparent production of Okazaki pieces from both daughter strands, DNA synthesis is actually asymmetric under these conditions. De novo chain initiation events occur on only one strand. It is suggested that asymmetry of synthesis in vivo may be masked by uracil excision and other postreplication processing mechanisms.
Full text
PDF




Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Dürwald H., Hoffmann-Berling H. DNA synthesis in nucleotide-permeable Escherichia coli cells. II. Synthesis of replicative from DNA of phage phi X174. J Mol Biol. 1971 Jun 28;58(3):755–773. doi: 10.1016/0022-2836(71)90038-6. [DOI] [PubMed] [Google Scholar]
- Eisenberg S., Scott J. F., Kornberg A. Enzymatic replication of viral and complementary strands of duplex DNA of phage phiX174 proceeds by seprate mechanisms. Proc Natl Acad Sci U S A. 1976 Sep;73(9):3151–3155. doi: 10.1073/pnas.73.9.3151. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Geider K. DNA synthesis in nucleotide-permeable Escherichia coli cells. The effects of nucleotide analogues on DNA synthesis. Eur J Biochem. 1972 Jun 9;27(3):554–563. doi: 10.1111/j.1432-1033.1972.tb01872.x. [DOI] [PubMed] [Google Scholar]
- Gottesman M. M., Hicks M. L., Gellert M. Genetics and function of DNA ligase in Escherichia coli. J Mol Biol. 1973 Jul 15;77(4):531–547. doi: 10.1016/0022-2836(73)90221-0. [DOI] [PubMed] [Google Scholar]
- Herrmann R., Huf J., Bonhoeffer F. Cross hybridization and rate of chain elongation of the two classes of DNA intermediates. Nat New Biol. 1972 Dec 20;240(103):235–237. doi: 10.1038/newbio240235a0. [DOI] [PubMed] [Google Scholar]
- Konrad E. B., Lehman I. R. Novel mutants of Escherichia coli that accumulate very small DNA replicative intermediates. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2150–2154. doi: 10.1073/pnas.72.6.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Konrad E. B., Modrich P., Lehman I. R. Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase. J Mol Biol. 1973 Jul 15;77(4):519–529. doi: 10.1016/0022-2836(73)90220-9. [DOI] [PubMed] [Google Scholar]
- Kuempel P. L., Veomett G. E. A possible function of DNA polymerase in chromosome replication. Biochem Biophys Res Commun. 1970 Nov 25;41(4):973–980. doi: 10.1016/0006-291x(70)90180-4. [DOI] [PubMed] [Google Scholar]
- Lark K. G. Genetic control over the initiation of the synthesis of the short deoxynucleotide chains in E. coli. Nat New Biol. 1972 Dec 20;240(103):237–240. doi: 10.1038/newbio240237a0. [DOI] [PubMed] [Google Scholar]
- Lindahl T. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3649–3653. doi: 10.1073/pnas.71.9.3649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okazaki R., Arisawa M., Sugino A. Slow joining of newly replicated DNA chains in DNA polymerase I-deficient Escherichia coli mutants. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2954–2957. doi: 10.1073/pnas.68.12.2954. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okazaki R., Okazaki T., Sakabe K., Sugimoto K., Sugino A. Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc Natl Acad Sci U S A. 1968 Feb;59(2):598–605. doi: 10.1073/pnas.59.2.598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Olivera B. M., Bonhoeffer F. Discontinuous DNA replication in vitro. I. Two distinct size classes of intermediates. Nat New Biol. 1972 Dec 20;240(103):233–235. doi: 10.1038/newbio240233a0. [DOI] [PubMed] [Google Scholar]
- Olivera R. M., Bonhoeffer E. Replication of Escherichia coli requires DNA polymerase I. Nature. 1974 Aug 9;250(5466):513–514. doi: 10.1038/250513a0. [DOI] [PubMed] [Google Scholar]
- Schaller H., Otto B., Nüsslein V., Huf J., Herrmann R., Bonhoeffer F. Deoxyribonucleic acid replication in vitro. J Mol Biol. 1972 Jan 28;63(2):183–200. doi: 10.1016/0022-2836(72)90369-5. [DOI] [PubMed] [Google Scholar]
- Schekman R., Weiner A., Kornberg A. Multienzyme systems of DNA replication. Science. 1974 Dec 13;186(4168):987–993. doi: 10.1126/science.186.4168.987. [DOI] [PubMed] [Google Scholar]
- Sternglanz R., Wang H. F., Donegan J. J. Evidence that both growing DNA chains at a replication fork are synthesized discontinuously. Biochemistry. 1976 May 4;15(9):1838–1843. doi: 10.1021/bi00654a008. [DOI] [PubMed] [Google Scholar]
- Tye B. K., Chien J., Lehman I. R., Duncan B. K., Warner H. R. Uracil incorporation: a source of pulse-labeled DNA fragments in the replication of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1978 Jan;75(1):233–237. doi: 10.1073/pnas.75.1.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tye B. K., Nyman P. O., Lehman I. R., Hochhauser S., Weiss B. Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA. Proc Natl Acad Sci U S A. 1977 Jan;74(1):154–157. doi: 10.1073/pnas.74.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uyemura D., Eichler D. C., Lehman I. R. Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. II. The polAex1 mutation. J Biol Chem. 1976 Jul 10;251(13):4085–4089. [PubMed] [Google Scholar]
- Wickner S., Hurwitz J. Conversion of phiX174 viral DNA to double-stranded form by purified Escherichia coli proteins. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4120–4124. doi: 10.1073/pnas.71.10.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
