scholarly journals Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

10.3791/55312 ◽  
2017 ◽  
Author(s):  
Alfredo J. Hernandez ◽  
Charles C. Richardson
Keyword(s):  
Dna Synthesis ◽  
Bacteriophage T7 ◽  
Replication Proteins ◽  
Kinetics Of ◽  
Lagging Strand

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins

2002 ◽  
Vol 316 (1) ◽  
pp. 19-34 ◽  
Author(s):  
Joonsoo Lee ◽  
Paul D Chastain ◽  
Jack D Griffith ◽  
Charles C Richardson
Keyword(s):  
Dna Synthesis ◽  
Bacteriophage T7 ◽  
Replication Proteins ◽  
Lagging Strand

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

Kinetics of hepatocyte growth factor-induced DNA synthesis in vitro

1993 ◽  
Vol 5 (6) ◽  
pp. 451-456
Author(s):  
Anthony C. Woodman ◽  
Jane Collard ◽  
Clare Selden ◽  
Humphrey J.F. Hodgson
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Dna Synthesis ◽  
Kinetics Of ◽  
Hepatocyte Growth

Studies on bacteriophage T7 DNA synthesis in vitro

1975 ◽  
Vol 141 (3) ◽  
pp. 233-249 ◽  
Author(s):  
Eberhard Scherzinger ◽  
Günther Klotz
Keyword(s):  
Dna Synthesis ◽  
Bacteriophage T7

scholarly journals Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

2000 ◽  
Vol 182 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Ying-Ta Lai ◽  
Warren Masker
Keyword(s):  
Dna Synthesis ◽  
High Efficiency ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Bacteriophage T7 ◽  
Growth Step ◽  
Strand Breaks

ABSTRACT An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5′→3′ exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.

In vitro concatemerization of bacteriophage T7 DNA: Role of DNA synthesis and gene 6 exonuclease

1985 ◽  
Vol 63 (4) ◽  
pp. 237-242 ◽  
Author(s):  
Donald D. Lee ◽  
Paul D. Sadowski
Keyword(s):  
Infected Cell ◽  
Dna Synthesis ◽  
Base Pair ◽  
Bacteriophage T7 ◽  
Cell Extracts ◽  
In Vitro Incubation ◽  
Insight Into

The replication of bacteriophage T7 DNA in vivo proceeds via the synthesis of complex concatemeric intermediates which are joined via the 160 base pair terminal redundancies at either end of the phage chromosome. To gain some insight into the mode of generation of these structures, we have examined the role of DNA synthesis in the formation of concatemeric bacteriophage T7 DNA in vitro. Incubation of mature T7 DNA with T7-infected cell extracts and a deoxynucleoside [32P]triphosphate resulted in the incorporation of significant radioactivity into the DNA. Highest levels of incorporation were at the termini of the DNA and decreased toward the middle of the molecule. Incorporation was dependent upon the presence of the activity of the gene 6 exonuclease and correlated with the generation of concatemeric DNA. A model explaining the role of exonucleolytic degradation and DNA synthesis in the generation of concatemeric DNA is presented.

scholarly journals Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.

1982 ◽  
Vol 79 (23) ◽  
pp. 7440-7444 ◽  
Author(s):  
L. A. Dodson ◽  
R. S. Foote ◽  
S. Mitra ◽  
W. E. Masker
Keyword(s):  
Dna Synthesis ◽  
Bacteriophage T7

Discontinuous leading-strand synthesis: a stop–start story

2014 ◽  
Vol 42 (1) ◽  
pp. 25-34 ◽  
Author(s):  
Joseph T.P. Yeeles
Keyword(s):  
Model Organisms ◽  
Replication Proteins ◽  
Lesion Bypass ◽  
Exogenous Dna ◽  
Dna Lesion ◽  
Dna Damaging Agents ◽  
Leading Strand ◽  
Lagging Strand

Reconstitution experiments using replication proteins from a number of different model organisms have firmly established that, in vitro, DNA replication is semi-discontinuous: continuous on the leading strand and discontinuous on the lagging strand. The mechanism by which DNA is replicated in vivo is less clear. In fact, there have been many observations of discontinuous replication in the absence of exogenous DNA-damaging agents. It has also been proposed that replication is discontinuous on the leading strand at least in part because of DNA lesion bypass. Several recent studies have revealed mechanistic details of pathways where replication of the leading strand introduces discontinuities. These mechanisms and their potential contributions to observations of discontinuous replication in vivo will be discussed.

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