Faculty Opinions recommendation of The replication intermediates in Escherichia coli are not the product of DNA processing or uracil excision.

2006 ◽  
Author(s):  
Martin Marinus
Keyword(s):  
Escherichia Coli ◽  
Replication Intermediates ◽  
Dna Processing

scholarly journals NikAB- or NikB-Dependent Intracellular Recombination between Tandemly Repeated oriT Sequences of Plasmid R64 in Plasmid or Single-Stranded Phage Vectors

2003 ◽  
Vol 185 (13) ◽  
pp. 3871-3877 ◽  
Author(s):  
Nobuhisa Furuya ◽  
Teruya Komano
Keyword(s):  
Escherichia Coli ◽  
Dna Transfer ◽  
The Other ◽  
E Coli ◽  
Core Sequence ◽  
Bacterial Plasmid ◽  
Plasmid R64 ◽  
Dna Processing ◽  
Origin Of Transfer

ABSTRACT The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.

scholarly journals Mechanisms of Transcription-Replication Collisions in Bacteria

2005 ◽  
Vol 25 (3) ◽  
pp. 888-895 ◽  
Author(s):  
Ekaterina V. Mirkin ◽  
Sergei M. Mirkin
Keyword(s):  
Escherichia Coli ◽  
Direct Contact ◽  
Replication Origin ◽  
Replication Fork ◽  
Mutual Orientation ◽  
Physical Interaction ◽  
Inducible Promoters ◽  
Replication Fork Progression ◽  
Replication Intermediates

ABSTRACT While collisions between replication and transcription in bacteria are deemed inevitable, the fine details of the interplay between the two machineries are poorly understood. In this study, we evaluate the effects of transcription on the replication fork progression in vivo, by using electrophoresis analysis of replication intermediates. Studying Escherichia coli plasmids, which carry constitutive or inducible promoters in different orientations relative to the replication origin, we show that the mutual orientation of the two processes determines their mode of interaction. Replication elongation appears not to be affected by transcription proceeding in the codirectional orientation. Head-on transcription, by contrast, leads to severe inhibition of the replication fork progression. Furthermore, we evaluate the mechanism of this inhibition by limiting the area of direct contact between the two machineries. We observe that replication pausing zones coincide exactly with transcribed DNA segments. We conclude, therefore, that the replication fork is most likely attenuated upon direct physical interaction with the head-on transcription machinery.

scholarly journals IncP Plasmids Are Unusually Effective in Mediating Conjugation of Escherichia coli and Saccharomyces cerevisiae: Involvement of the Tra2 Mating System

1998 ◽  
Vol 180 (24) ◽  
pp. 6538-6543 ◽  
Author(s):  
Steven Bates ◽  
Annette M. Cashmore ◽  
Brian M. Wilkins
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Gene Transfer ◽  
Recipient Cell ◽  
Processing System ◽  
Conjugation System ◽  
Shuttle Plasmid ◽  
K 12 ◽  
Core Genes ◽  
Dna Processing

ABSTRACT Mobilizable shuttle plasmids containing the origin-of-transfer (oriT) region of plasmids F (IncFI), ColIb-P9 (IncI1), and RP4/RP1 (IncPα) were constructed to test the ability of the cognate conjugation system to mediate gene transfer from Escherichia coli to Saccharomyces cerevisiae. Only the Pα system caused detectable mobilization to yeast, giving peak values of 5 × 10−5 transconjugants per recipient cell in 30 min. Transfer of the shuttle plasmid required carriage oforiT in cis and the provision intrans of the Pα Tra1 core and Tra2 core regions. Genes outside the Tra1 core did not increase the mobilization efficiency. All 10 Tra2 core genes (trbB, -C, -D, -E, -F, -G, -H, -I, -J, and -L) required for plasmid transfer to E. coli K-12 were needed for transfer to yeast. To assess whether the mating-pair formation (Mpf) system or DNA-processing apparatus of the Pα conjugation system is critical in transkingdom transfer, an assay using an IncQ-based shuttle plasmid specifying its own DNA-processing system was devised. RP1 but not ColIb mobilized the construct to yeast, indicating that the Mpf complex determined by the Tra2 core genes plus traF is primarily responsible for the remarkable fertility of the Pα system in mediating gene transfer from bacteria to eukaryotes.

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid pBR322

Genetics ◽  
1987 ◽  
Vol 115 (1) ◽  
pp. 41-49
Author(s):  
Ujjala DasGupta ◽  
Kathleen Weston-Hafer ◽  
Douglas E Berg
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Specific Interactions ◽  
Plasmid Pbr322 ◽  
Direct Repeats ◽  
Ampicillin Resistance ◽  
Deletion Formation ◽  
Coli Plasmid ◽  
Insertion Mutations ◽  
Dna Processing

ABSTRACT The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.

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