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Exodeoxyribonuclease I (EC 3.1.11.1, Escherichia coli exonuclease I, E. coli exonuclease I, exonuclease I) is an enzyme  that catalyses the following chemical reaction:


 * Exonucleolytic cleavage in the 3′- to 5′-direction to yield nucleoside 5′-phosphates and has been implicated in DNA recombination and repair.

Preference for single-stranded DNA. The Escherichia coli enzyme hydrolyses glucosylated DNA.

EC number of the enzyme
The EC number of Exodeoxyribonuclease I is 3.1.11.1. The first number 3 means that it is in class 3 which is hydrolases enzymes. EC 3.1 are the esterases. EC 3.1.11-31 are nucleases including deoxyribonuclease (DNase) and ribonuclease (RNase). EC 3.1.11-16 are exonucleases and EC 3.1.21-31 are endonucleases. EC 3.1.11 is exodeoxyribonucleases producing 5’-phosphomonoesters. The serial number is 1.

Reaction pathway
Exodeoxyribonuclease I conduct exonucleolytic cleavage in the direction of 3' to 5' and yield nucleoside 5'-phosphates. It hydrolyzes single stranded DNA via a metal catalytic mechanism which requires two divalent metal ions, Mg2+. Two metal ions are in the active site, establishing a pattern for phosphodiester bond hydrolysis. One of the divalent ion is presumed to initiate the hydrolysis of phosphodiester bond between DNA by forming the attacking hydroxide ion; the other stabilizes the pentacoordinated transition state and/or oxyanion leaving-group.

Organism
Exonuclease I (ExoI) is found in Escherichia coli . ExoI seems to be discovered only in gamma-proteobacteria and not widespread.

Enzyme function (in cell)
Exonuclease I is reported to be involved in the process of DNA genetic recombination, mutation avoidance, and repair in E. coli. In bacteria, the lesions of single-stranded DNA and double-stranded DNA might happen due to the contact between the chromosome and cytoplasm, radicals, and other chemicals. Therefore, they evolve the DNA repair mechanisms to resist these reactions. In the beginning, Exonuclease I (ExoI) was recognized as the suppressor of recombination in recBC mutants, yet the subsequent research states that the multiple exonuclease in E. coli play important roles in the recombination and repair pathways. The specific requirement of ExoI in apurinic or apyrimidinic sites repair suggests the possible effect on the correct alignment and suppression of mutations. Methyl-directed mismatch repair/correction (MMR) in E. coli requires the following protein components: MutHLS reaction (MutS, MutL, MutH) for activation of MutH endonuclease, four most potent exonucleases (ExoI, ExoVII, ExoX, and RecJ) , single-stranded DNA binding protein (SSB), DNA helicase II (MutU/UvrD), DNA polymerase III holoenzyme, and DNA ligase. The first step of DNA repair is MutS binding to the mismatch DNA (mostly ssDNA). Following that, MutL will bind to heteroduplex DNA which is dependent on MutS and ATP in order to stabilize the interaction and other proteins in the repair process. This complex can activate the endonuclease activity of MutH, cleaving the unmethylated strand of a hemimethylated sequence that can be on either side of the mispair DNA. Initial biochemical analysis suggests the involvement of the hydrolysis of exonuclease I in 3' to 5' orientation in mismatch repair process after the strand break occurs at 3’ to the mispair DNA caused by MutH.

Crystal structure
The crystal structure of ExoI without DNA is reported to have a C-shaped molecule of three domains. First is an N-terminal nuclease domain (residues 1–201) that has the same relative structure to the proofreading domain of E. coli DNA polymerase I, this domain also shows the similarity of structure to other DnaQ superfamily enzymes. Second part is the central domain that the part of portion shares similarity to the SH3 domain fold (residues 202–354). Last is the C-terminal α-helical domain (residues 359–475).

Active sites
The active site is at the bottom of the groove part. Four of the five conserved residues in the DnaQ superfamily within ExoI are acidic and can bind two metal ions. Comparing to the other protein in DnaQ superfamily, Klenow fragment (KF) which is the large protein fragment cleaved from DNA polymerase I of E. coli., ExoI substitutes the Tyr 497 with His 181 which is the presumed to help the alignment of the nucleotides for hydrolysis. According to the similarity, the hydrolysis of ExoI is assumed to happen by attacking the scissile phosphate with one activated water molecule. The extended loop region will enclose the DNA part and overlap the groove part. The 3’ end of ssDNA is bound in the active site and the downstream end under the crossover loop. The middle part of the DNA is going to form a bulge, which approach the SH3-like domain, and the downstream end of the DNA form extensive interactions with an anchor site.

Functional structure
In the middle of ExoI, there is a positive-charged groove with the nuclease active site at the bottom end. At the same site, one loop is bridging the SH3-like domain with C-terminal domains which overlaps through the top. The size of this groove can accommodate ∼12–13 nucleotides of ssDNA, which is compatible to the released product from previous literature.