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Structure of an Intermediate in the Replication of Bacteriophage φX174 Deoxyribonucleic Acid : The Initiation Site for DNA Replication

Johnson, Paul H. and Sinsheimer, Robert L. (1974) Structure of an Intermediate in the Replication of Bacteriophage φX174 Deoxyribonucleic Acid : The Initiation Site for DNA Replication. Journal of Molecular Biology, 83 (1). pp. 47-61. ISSN 0022-2836. https://resolver.caltech.edu/CaltechAUTHORS:20160805-105728591

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Abstract

Replicative form DNA composed of a closed complementary strand and a discontinuous viral strand has been isolated from cells infected with bacteriophage φX174 during the period of single-strand DNA synthesis. This RFII DNA was degraded by the restriction enzyme from Hemophilus influenzae, endonuclease R, and the products analyzed by polyacrylamide gel electrophoresis. The results indicate that there are two types of discontinuity in the viral strands of these molecules: (1) 65% of the molecules contain a gap, which causes a discrete increase in mobility of a specific restriction enzyme fragment, R3. This gap can be selectively repaired with Escherichia coli DNA polymerase I and nucleoside triphosphates, but the molecules are not converted to RFI by addition of E. coli polynueleotide ligase to the reaction mixture. Approximately 30 moles of radioactive TTP are incorporated per mole of RF DNA. (2) 35% of the RF molecules contain a discontinuity, which does not result in a detectable change in mobility of any restriction enzyme fragment. These RF molecules can be converted to RFI by the action of ligase and polymerase I in the presence of nucleoside triphosphates, with incorporation of only approximately one mole of radioactive TTP, specifically into fragment R3, per mole of RF DNA. When the reaction of late RFII DNA and polymerase I is allowed to proceed beyond the repair of the discontinuity, radioactive nucleotides are incorporated into endonuclease R fragments adjacent to R3 in the 5′ → 3′ direction. This technique was utilized to determine a partial order of endonuclease R fragments in φX174. These results suggest that the synthesis of single-strand DNA is initiated from a unique point in cistron A and proceeds clockwise round the φX174 genetic map (cistron order: ABCDEFGH). A comparison of these results with other studies on φX174 suggests that DNA synthesis in all stages of φX174 replication may be initiated from a specific locus on the genome, at or near cistron A.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/0022-2836(74)90423-9DOIArticle
http://www.sciencedirect.com/science/article/pii/0022283674904239PublisherArticle
Additional Information:© 1974 Elsevier Ltd. Received 21 May 1973. This research was supported by grant no. GM13554 from the U.S. Public Health Service. One of us (P. H. J.) was supported by a postdoctoral research fellowship no. PF664 from the American Cancer Society.
Funders:
Funding AgencyGrant Number
U.S. Public Health Service (USPHS)GM 13554
American Cancer SocietyPF664
Issue or Number:1
Record Number:CaltechAUTHORS:20160805-105728591
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160805-105728591
Official Citation:Paul H. Johnson, Robert L. Sinsheimer, Structure of an intermediate in the replication of bacteriophage φX174 deoxyribonucleic acid: The initiation site for DNA replication, Journal of Molecular Biology, Volume 83, Issue 1, 15 February 1974, Pages 47-61, ISSN 0022-2836, http://dx.doi.org/10.1016/0022-2836(74)90423-9. (http://www.sciencedirect.com/science/article/pii/0022283674904239)
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ID Code:69473
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:05 Aug 2016 18:10
Last Modified:03 Oct 2019 10:22

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