Oct 26, 2018

Exonuclease III (XthA) enforces in vivo DNA cloning of Escherichia coli to create cohesive ends

BioRxiv : the Preprint Server for Biology
Shingo Nozaki, Hironori Niki

Abstract

Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-cloning technology. However, the molecular mechanism by which E. coli accomplishes such cloning is still unknown. In this study, we provide evidence that the in vivo cloning of E. coli is independent of both RecA and RecET recombinase, but is dependent on XthA, a 3' to 5' exonuclease. Here, in vivo cloning of E. coli by XthAis referred to as iVEC (in vivo E. coli cloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3' ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5' overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA-cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to s...Continue Reading

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Mentioned in this Paper

Pola Office
Study
In Vivo
Rec A Recombinases
Carboxy-Terminal Amino Acid
DNA Repair
Gene Deletion Abnormality
Gene Deletion
Phosphodiesterase I
Recombinase

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