title: Rpn (YhgA-Like) Proteins of Escherichia coli K-12 and Their Contribution to RecA-Independent Horizontal Transfer.
abstract: Bacteria use a variety of DNA-mobilizing enzymes to facilitate environmental niche adaptation via horizontal gene transfer. This has led to real-world problems, like the spread of antibiotic resistance, yet many mobilization proteins remain undefined. In the study described here, we investigated the uncharacterized family of YhgA-like transposase_31 (Pfam PF04754) proteins. Our primary focus was the genetic and biochemical properties of the fiveEscherichia coliK-12 members of this family, which we designate RpnA to RpnE, where Rpn representsrecombination-promotingnuclease. We employed a conjugal system developed by our lab that demanded RecA-independent recombination following transfer of chromosomal DNA. Overexpression of RpnA (YhgA), RpnB (YfcI), RpnC (YadD), and RpnD (YjiP) increased RecA-independent recombination, reduced cell viability, and induced the expression of reporter of DNA damage. For the exemplar of the family, RpnA, mutational changes in proposed catalytic residues reduced or abolished all three phenotypes in concert.In vitro, RpnA displayed magnesium-dependent, calcium-stimulated DNA endonuclease activity with little, if any, sequence specificity and a preference for double-strand cleavage. We propose that Rpn/YhgA-like family nucleases can participate in gene acquisition processes.IMPORTANCEBacteria adapt to new environments by obtaining new genes from other bacteria. Here, we characterize a set of genes that can promote the acquisition process by a novel mechanism. Genome comparisons had suggested the horizontal spread of the genes for the YhgA-like family of proteins through bacteria. Although annotated as transposase_31, no member of the family has previously been characterized experimentally. We show that fourEscherichia coliK-12 paralogs contribute to a novel RecA-independent recombination mechanismin vivoFor RpnA, we demonstratein vitroaction as a magnesium-dependent, calcium-stimulated nonspecific DNA endonuclease. The cleavage products are capable of providing priming sites for DNA polymerase, which can enable DNA joining by primer-template switching.
authors.affiliation: New England BioLabs, Ipswich, Massachusetts, USA.
authors.forename: Anthony W
authors.identifier: http://orcid.org/0000-0002-5560-2880
authors.initials: AW
authors.lastname: Kingston
authors.affiliation: New England BioLabs, Ipswich, Massachusetts, USA.
authors.forename: Christine
authors.identifier: http://orcid.org/0000-0001-6776-4556
authors.initials: C
authors.lastname: Ponkratz
authors.affiliation: New England BioLabs, Ipswich, Massachusetts, USA raleigh@neb.com.
authors.forename: Elisabeth A
authors.identifier: http://orcid.org/0000-0003-0745-9925
authors.initials: EA
authors.lastname: Raleigh
mesh_terms: D002118:Calcium / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D022761:Gene Transfer, Horizontal / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D021381:Protein Transport; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
chemical_list: D029968:Escherichia coli Proteins; D011938:Rec A Recombinases; D003850:Deoxyribonuclease I; D008274:Magnesium; D002118:Calcium
keywords: DNA damage; conjugation; evolutionary biology; gene function; genomic instability; horizontal gene transfer; illegitimate recombination; protein function; protein motif; transposase