Protein-primed replication of the bacteriophage PRD1 genome in vitro: Development of in vitro DNA replication system and characterization of replication origin.

Persistent Link:
http://hdl.handle.net/10150/185299
Title:
Protein-primed replication of the bacteriophage PRD1 genome in vitro: Development of in vitro DNA replication system and characterization of replication origin.
Author:
Yoo, Seung-Ku.
Issue Date:
1990
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
A cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 (DNA polymerase) and 8 (terminal protein) of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-terminal protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. The origin and direction of PRD1 DNA replication in vitro were determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at either end of the linear PRD1 DNA template. Analysis by alkaline sucrose gradient centrifugation and alkaline agarose gel electrophoresis of DNA synthesized in vitro showed that full-length PRD1 DNA is synthesized. DNA elongation in this system is inhibited by the drug aphidicolin. On the other hand, DNA initiation is inhibited by phenylglyoxal, an arginine-specific α-dicarbonyl reagent. In vitro studies have also demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. The minimal origin of replication was determined by analyzing the template activity of various deletion derivatives. It was shown that the terminal 20 bp of ITR are required for efficient in vitro DNA replication. It was found that, within the minimal replication origin region, there are complementary sequences which can form a small panhandle structure. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3' terminal C residue for the initiation of PRD1 DNA replication in vitro.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology and Immunology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Ito, Junetsu

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleProtein-primed replication of the bacteriophage PRD1 genome in vitro: Development of in vitro DNA replication system and characterization of replication origin.en_US
dc.creatorYoo, Seung-Ku.en_US
dc.contributor.authorYoo, Seung-Ku.en_US
dc.date.issued1990en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractA cell-free system has been developed from cells of an Escherichia coli strain, carrying cloned genes 1 (DNA polymerase) and 8 (terminal protein) of bacteriophage PRD1, that catalyzes protein-primed DNA synthesis. DNA synthesis in vitro is entirely dependent upon the addition of PRD1 DNA-terminal protein complex as template, Mg²⁺, and four deoxyribonucleoside triphosphates. The origin and direction of PRD1 DNA replication in vitro were determined by restriction enzyme analysis of ³²P-labeled PRD1 DNA synthesized in this system. Replication starts at either end of the linear PRD1 DNA template. Analysis by alkaline sucrose gradient centrifugation and alkaline agarose gel electrophoresis of DNA synthesized in vitro showed that full-length PRD1 DNA is synthesized. DNA elongation in this system is inhibited by the drug aphidicolin. On the other hand, DNA initiation is inhibited by phenylglyoxal, an arginine-specific α-dicarbonyl reagent. In vitro studies have also demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. The minimal origin of replication was determined by analyzing the template activity of various deletion derivatives. It was shown that the terminal 20 bp of ITR are required for efficient in vitro DNA replication. It was found that, within the minimal replication origin region, there are complementary sequences which can form a small panhandle structure. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3' terminal C residue for the initiation of PRD1 DNA replication in vitro.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiologyen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorIto, Junetsuen_US
dc.contributor.committeememberLittle, Johnen_US
dc.contributor.committeememberBernstein, Carolen_US
dc.contributor.committeememberHall, Jenniferen_US
dc.contributor.committeememberSpizizen, Johnen_US
dc.identifier.proquest9111984en_US
dc.identifier.oclc710837405en_US
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