The enterohemorrhagicEscherichia coliinsertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity

Abstract

Bacterial genomes contain an abundance of transposable insertion sequence (IS) elements that are essential for genome evolution and fitness. Among them, IS629 is present in most strains of enterohemorrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inactivation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identified in the most pathogenic serotypes of E. coli, its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome assemblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and enterotoxigenic genomes. In vitro analysis of the recombinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA polymerase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efficiently perform in vitro an end-joining reaction of 3’-single-strand DNA overhangs with ≥4 bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.

The insertion sequence excision enhancer: A PrimPol‐based primer invasion system for immobilizing transposon‐transmitted antibiotic resistance genes

“…Indeed, as proposed from alignments with other members of this protein family (Calvo et al, 2023;Kusumoto et al, 2011; Figure 3),…”

“…biochemical analyses using purified IEE demonstrated that the enzyme possesses DNA polymerase activity in the presence of Mg 2+ (Calvo et al, 2023) and can extend a 15 bp primer on a short 33 bp template using dNTPs but not NTPs (Figure 4ai). Mutation of two of the probable AEP catalytic residues (Figure 3), D142A/D144A, eliminated this activity.…”

“…Using appropriately 5′ resected double-strand oligonucleotides (Figure 4b) with 0, 2, 4, and 6 nucleotide microhomologies at their 3′ ends (Figure 4b A polymerization reaction including the four dNTPs revealed that IEE could specifically elongate a significant proportion of the 4 and 6 nucleotide substrates (28 and 46%) although no detectable elongation was observed with the 2 nt substrate under the in vitro reaction conditions used. IEE was also capable of limited synapsing of single strand substrates with 3′ terminal microhomologies (Calvo et al, 2023).…”

“…The authors propose that it might even contribute to the removal of transposase from the transposition complex (Calvo et al, 2023). 2023).…”

“…More recently, it has been estimated that a highly conserved gene copy is present in over 30% of available E. coli genome assemblies and is very abundant not only within enterohemorrhagic and enterotoxigenic genomes but also within enteropathogenic E. coli in general (Calvo et al, 2023). The ECs1305 gene was subsequently named iee for I S‐ e xcision e nhancer (Kusumoto et al, 2011).…”