scholarly journals An 18-base-pair sequence is sufficient for termination of rolling-circle replication of plasmid pT181.

1996 ◽  
Vol 178 (17) ◽  
pp. 5222-5228 ◽  
Author(s):  
A C Zhao ◽  
S A Khan
Keyword(s):  
Base Pair ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Pair Sequence ◽  
Plasmid Pt181

scholarly journals Dynamics of DNA nicking and unwinding by the RepC–PcrA complex

2020 ◽  
Vol 48 (4) ◽  
pp. 2013-2025 ◽  
Author(s):  
Carolina Carrasco ◽  
Cesar L Pastrana ◽  
Clara Aicart-Ramos ◽  
Sanford H Leuba ◽  
Saleem A Khan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Antibiotic Resistance Genes ◽  
Dna Supercoiling ◽  
Magnetic Tweezers ◽  
Common Mechanism ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Gram Positive Bacteria ◽  
Dna Nicking ◽  
Plasmid Pt181

Abstract The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s−1, while a typical velocity of 50 bp s−1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

scholarly journals Targeted Allele Replacement Mutagenesis of Corynebacterium pseudotuberculosis

2011 ◽  
Vol 77 (10) ◽  
pp. 3532-3535 ◽  
Author(s):  
Caray A. Walker ◽  
Willie Donachie ◽  
David G. E. Smith ◽  
Michael C. Fontaine
Keyword(s):  
Corynebacterium Pseudotuberculosis ◽  
Sequence Length ◽  
Homologous Sequence ◽  
Proof Of Concept ◽  
Rolling Circle Replication ◽  
Content Type ◽  
Rolling Circle ◽  
Allele Replacement ◽  
Marker Free ◽  
The Relationship

ABSTRACTA two-step allele replacement mutagenesis procedure, using a conditionally replicating plasmid, was developed to allow the creation of targeted, marker-free mutations inCorynebacterium pseudotuberculosis. The relationship between homologous sequence length and recombination frequency was determined, and enhanced plasmid excision was observed due to the rolling-circle replication of the mutagenesis vector. Furthermore, an antibiotic enrichment procedure was applied to improve the recovery of mutants. Subsequently, as proof of concept, a marker-free,cp40-deficient mutant ofC. pseudotuberculosiswas constructed.

Homogeneous Detection of Single Rolling Circle Replication Products

2004 ◽  
Vol 76 (2) ◽  
pp. 495-498 ◽  
Author(s):  
Gerhard A. Blab ◽  
Thomas Schmidt ◽  
Mats Nilsson
Keyword(s):  
Rolling Circle Replication ◽  
Rolling Circle

scholarly journals Genetic analysis of the human thymidine kinase gene promoter.

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909 ◽  
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

Genetic analysis of the human thymidine kinase gene promoter

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

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