Literature summary for 3.1.11.2 extracted from

Nozaki, S.; Niki, H.
Exonuclease III (XthA) enforces in vivo DNA cloning of Escherichia coli to create cohesive ends (2019), J. Bacteriol., 201, e00660-18 .

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Application

Application	Comment	Organism
molecular biology	in contrast to DNA cloning utilizing in vitro recombination, some strains of Escherichia coli can take up linear double-stranded vectors, insert DNA fragments, and assemble them in vivo involving exonulcease III. The ends of these linear DNA fragments must contain 20 to 50 bp of overlapping homologous sequences. Improved protocols for in vivo cloning have realized a high level of usability comparable to that by in vitro recombination reactions, but using the exonuclease III, it is only necessary to introduce PCR products into Escherichia coli for the in vivo cloning	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
additional information	elucidation of the iVEC mechanism at the molecular level avances the development of in vivo DNA cloning technology. Multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC is possible. In vitro recombination system for DNA cloning already allow the joining of multiple DNA fragments at once. Construction of an Escherichia coli strain that is optimized for in vivo cloning. Generation of exonuclease deletion mutants from strain BW25113, the parental strain of the Keio collection, has sufficient capacity for iVEC activity	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
additional information	Escherichia coli	XthA exonuclease converts the blunt ends of double-stranded DNA to 5' protruding ends in the process of in vivo cloning. After the insert and the vector DNA fragments are introduced into the Escherichia coli cell, XthA resects the ends of the DNA fragments from the 3'-to-5' direction, producing 5' overhanging ends. As the ends of insert and vector DNAs have mutually complementary sequences, the 5' overhanging ends of the insert and the vector DNA fragments hybridize to each other as cohesive ends. In addition, the gaps are filled by DNA polymerases and the nicks are repaired by DNA ligases	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P09030	-	-

Reaction

Reaction	Comment	Organism	Reaction ID
Exonucleolytic cleavage in the 3'- to 5'- direction to yield nucleoside 5'-phosphates	XthA exonuclease converts the blunt ends of double-stranded DNA to 5' protruding ends in the process of in vivo cloning. After the insert and the vector DNA fragments are introduced into the Escherichia coli cell, XthA resects the ends of the DNA fragments from the 3'-to-5' direction, producing 5' overhanging ends. As the ends of insert and vector DNAs have mutually complementary sequences, the 5' overhanging ends of the insert and the vector DNA fragments hybridize to each other as cohesive ends. In addition, the gaps are filled by DNA polymerases and the nicks are repaired by DNA ligases	Escherichia coli	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	XthA exonuclease converts the blunt ends of double-stranded DNA to 5' protruding ends in the process of in vivo cloning. After the insert and the vector DNA fragments are introduced into the Escherichia coli cell, XthA resects the ends of the DNA fragments from the 3'-to-5' direction, producing 5' overhanging ends. As the ends of insert and vector DNAs have mutually complementary sequences, the 5' overhanging ends of the insert and the vector DNA fragments hybridize to each other as cohesive ends. In addition, the gaps are filled by DNA polymerases and the nicks are repaired by DNA ligases	Escherichia coli	?	-	?
additional information	XthA, also known as exodeoxyribonuclease III, exhibits 3' to 5' exonuclease activity. The sbcA23 mutant of the Escherichia coli strain JC8679 is used for in vivo cloning, because the expression of RecE exonuclease and RecT recombinase of Rac prophage is activated in this mutant. Multiple fragment cloning by the host strain SN1187. The cat fragment and linearized pUC19 are used for the transformation of indicated strains	Escherichia coli	?	-	?

Synonyms

Synonyms	Comment	Organism
exonuclease III	-	Escherichia coli
xthA	-	Escherichia coli

General Information

General Information	Comment	Organism
evolution	in general, DNA recombination in Escherichia coli accompanies the conversion of double-stranded DNA to single-stranded DNA by exonuclease. It is reported that Escherichia coli has at least seven exonucleases that prefer double-stranded DNA for their substrates: XthA, RecE, ExoX, RecBCD, SbcCD, Nfo, and TatD. In addition, YgdG is an exonuclease with yet undetermined substrate preference. It seems likely that Escherichia coli K-12 originally acquired iVEC activity, and the iVEC activity was involved in an unknown physiological function in Escherichia coli	Escherichia coli
malfunction	deletion of the DNA polymerase domain of PolA does not completely abrogate iVEC activity	Escherichia coli
metabolism	involvement of DNA polymerases in in vivo cloning of Escherichia coli (iVEC) activity, analysis of iVEC activities of various strains, which are deletion mutants of nonessential polymerases in the Keio collection, overview. XthA plays a critical role in the iVEC activity	Escherichia coli
physiological function	Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. In vivo cloning of Escherichia coli (iVEC) is independent of both RecA and RecET recombinases but is dependent on XthA, a 3' to 5' exonuclease. XthA resects the 3' ends of linear DNA fragments that are introduced into Escherichia 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, molecular iVEC mechanism, overview. XthA helps to repair minor DNA damage, instead of the RecBCD exonuclease. RecBCD produces a 3' overhang and loads RecA onto the single-stranded DNA, causing an SOS response accompanied by cell division arrest. To help avoid such a serious outcome, it is conceivable that XthA functions in a repair pathway of DNA damage	Escherichia coli

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Release 2023.1 (January 2023)