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. 1971 Apr;11(4):355–369. doi: 10.1016/s0006-3495(71)86220-3

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

Ramendra K Poddar, Robert L Sinsheimer
PMCID: PMC1483972  PMID: 4931396

Abstract

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BOLLUM F. J., SETLOW R. B. Ultraviolet inactivation of DNA primer activity. I. Effects of different wavelengths and doses. Biochim Biophys Acta. 1963 Apr 30;68:599–607. doi: 10.1016/0006-3002(63)90189-6. [DOI] [PubMed] [Google Scholar]
  2. Burton A., Sinsheimer R. L. The process of infection with bacteriophage phi-X174 VII. Ultracentrifugal analysis of the replicative form. J Mol Biol. 1965 Dec;14(2):327–347. doi: 10.1016/s0022-2836(65)80185-1. [DOI] [PubMed] [Google Scholar]
  3. CASTELLANI A., JAGGER J., SETLOW R. B. OVERLAP OF PHOTOREACTIVATION AND LIQUID HOLDING RECOVERY IN ESCHERICHIA COLI B. Science. 1964 Mar 13;143(3611):1170–1171. doi: 10.1126/science.143.3611.1170. [DOI] [PubMed] [Google Scholar]
  4. Datta B., Poddar R. K. In vivo repair of UV-irradiation damage of single stranded DNA phage phi-X 174. Mol Gen Genet. 1970;107(1):50–57. doi: 10.1007/BF00433223. [DOI] [PubMed] [Google Scholar]
  5. Edgell M. H., Hutchison C. A., 3rd, Sinsheimer R. L. The process of infection with bacteriophage phi-X174. 28. Removal of the spike proteins from the phage capsid. J Mol Biol. 1969 Jun 28;42(3):547–557. doi: 10.1016/0022-2836(69)90242-3. [DOI] [PubMed] [Google Scholar]
  6. GUTHRIE G. D., SINSHEIMER R. L. Observations on the infection of bacterial protoplasts with the deoxyribonucleic acid of bacteriophage phi X174. Biochim Biophys Acta. 1963 Jun 25;72:290–297. [PubMed] [Google Scholar]
  7. Goulian M., Kornberg A. Enzymatic synthesis of DNA. 23. Synthesis of circular replicative form of phage phi-X174 DNA. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1723–1730. doi: 10.1073/pnas.58.4.1723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goulian M., Kornberg A., Sinsheimer R. L. Enzymatic synthesis of DNA, XXIV. Synthesis of infectious phage phi-X174 DNA. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2321–2328. doi: 10.1073/pnas.58.6.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. JOSSE J., KAISER A. D., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of nearest neighbor base sequences in deoxyribonucleic acid. J Biol Chem. 1961 Mar;236:864–875. [PubMed] [Google Scholar]
  10. Knippers R., Strätling W. The DNA replicating capacity of isolated E. coli cell wall-membrane complexes. Nature. 1970 May 23;226(5247):713–717. doi: 10.1038/226713a0. [DOI] [PubMed] [Google Scholar]
  11. Komano T., Sinsheimer R. L. Preparation and purification of phi X-RF component I. Biochim Biophys Acta. 1968 Jan 29;155(1):295–298. doi: 10.1016/0005-2787(68)90360-2. [DOI] [PubMed] [Google Scholar]
  12. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  13. Muhammed A. Studies on the yeast photoreactivating enzyme. I. A method for the large scale purification and some properties of the enzyme. J Biol Chem. 1966 Jan 25;241(2):516–523. [PubMed] [Google Scholar]
  14. ONO J., WILSON R. G., GROSSMAN L. EFFECTS OF ULTRAVIOLET LIGHT ON THE TEMPLATE PROPERTIES OF POLYCYTIDYLIC ACID. J Mol Biol. 1965 Mar;11:600–612. doi: 10.1016/s0022-2836(65)80014-6. [DOI] [PubMed] [Google Scholar]
  15. Pearson M. L., Ottensmeyer F. P., Johns H. E. Properties of an unusual photoproduct of U.V. irradiated thymidylyl-thymidine. Photochem Photobiol. 1965 Sep;4(4):739–747. doi: 10.1111/j.1751-1097.1965.tb07916.x. [DOI] [PubMed] [Google Scholar]
  16. RICHARDSON C. C., INMAN R. B., KORNBERG A. ENZYMIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID. 18. THE REPAIR OF PARTIALLY SINGLE-STRANDED DNA TEMPLATES BY DNA POLYMERASE. J Mol Biol. 1964 Jul;9:46–69. doi: 10.1016/s0022-2836(64)80090-5. [DOI] [PubMed] [Google Scholar]
  17. SETLOW R. B., CARRIER W. L., BOLLUM F. J. NUCLEASE-RESISTANT SEQUENCES IN ULTRAVIOLET-IRRADIATED DEOXYRIBONUCLEIC ACID. Biochim Biophys Acta. 1964 Nov 15;91:446–461. doi: 10.1016/0926-6550(64)90075-1. [DOI] [PubMed] [Google Scholar]
  18. SETLOW R. B., CARRIER W. L. THE DISAPPEARANCE OF THYMINE DIMERS FROM DNA: AN ERROR-CORRECTING MECHANISM. Proc Natl Acad Sci U S A. 1964 Feb;51:226–231. doi: 10.1073/pnas.51.2.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. STUDIER F. W. SEDIMENTATION STUDIES OF THE SIZE AND SHAPE OF DNA. J Mol Biol. 1965 Feb;11:373–390. doi: 10.1016/s0022-2836(65)80064-x. [DOI] [PubMed] [Google Scholar]
  20. Setlow J. K., Boling M. E., Bollum F. J. The chemical nature of photoreactivable lesions in DNA. Proc Natl Acad Sci U S A. 1965 Jun;53(6):1430–1436. doi: 10.1073/pnas.53.6.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Setlow R. B., Carrier W. L., Bollum F. J. Pyrimidine dimers in UV-irradiated poly dI:dC. Proc Natl Acad Sci U S A. 1965 May;53(5):1111–1118. doi: 10.1073/pnas.53.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tao M., Small G. D., Gordon M. P. Photochemical alterations in ribonucleic acid isolated from ultraviolet-irradiated tobacco mosaic virus. Virology. 1969 Nov;39(3):534–541. doi: 10.1016/0042-6822(69)90100-7. [DOI] [PubMed] [Google Scholar]
  23. WACKER A., DELLWEG H., JACHERTS D. [Thymine dimerization and survival of bacteria]. J Mol Biol. 1962 May;4:410–412. doi: 10.1016/s0022-2836(62)80022-9. [DOI] [PubMed] [Google Scholar]
  24. WULFF D. L., RUPERT C. S. Disappearance of thymine photodimer in ultraviolet irradiated DNA upon treatment with a photoreactivating enzyme from baker's yeast. Biochem Biophys Res Commun. 1962 Apr 20;7:237–240. doi: 10.1016/0006-291x(62)90181-x. [DOI] [PubMed] [Google Scholar]
  25. Wang S. Y., Varghese A. J. Cytosine-thymine addition product from DNA irradiated with ultraviolet light. Biochem Biophys Res Commun. 1967 Nov 30;29(4):543–549. doi: 10.1016/0006-291x(67)90519-0. [DOI] [PubMed] [Google Scholar]
  26. Yarus M., Sinsheimer R. L. Ultraviolet sensitivity of the biological activity of phi-X-174 virus, single-stranded DNA, and RF DNA. Biophys J. 1967 May;7(3):267–278. doi: 10.1016/S0006-3495(67)86587-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

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