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RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

ABSTRACT: Most, but not all, homologous genetic recombination in bacteria is mediated by the RecA recombinase. The mechanistic origin of RecA-independent recombination has remained enigmatic. Here, we demonstrate that the RarA protein makes a major enzymatic contribution to RecA-independent recombination. In particular, RarA makes substantial contributions to intermolecular recombination and to recombination events involving relatively short (<200 bp) homologous sequences, where RecA-mediated recombination is inefficient. The effects are seen here in plasmid-based recombination assays and in vivo cloning processes. Vestigial levels of recombination remain even when both RecA and RarA are absent. Additional pathways for RecA-independent recombination, possibly mediated by helicases, are suppressed by exonucleases ExoI and RecJ. Translesion DNA polymerases may also contribute. Our results provide additional substance to a previous report of a functional overlap between RecA and RarA.

SUBMITTER: Jain K

PROVIDER: S-EPMC8160026 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

Jain Kanika K Wood Elizabeth A EA Romero Zachary J ZJ Cox Michael M MM

Molecular microbiology 20201219 6

Most, but not all, homologous genetic recombination in bacteria is mediated by the RecA recombinase. The mechanistic origin of RecA-independent recombination has remained enigmatic. Here, we demonstrate that the RarA protein makes a major enzymatic contribution to RecA-independent recombination. In particular, RarA makes substantial contributions to intermolecular recombination and to recombination events involving relatively short (<200 bp) homologous sequences, where RecA-mediated recombinatio ...[more]

PMID: 33247976

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Project description: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 five Escherichia coli K-12 members of this family, which we designate RpnA to RpnE, where Rpn represents recombination-promoting nuclease. 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.IMPORTANCE Bacteria 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 four Escherichia coli K-12 paralogs contribute to a novel RecA-independent recombination mechanism in vivo For RpnA, we demonstrate in vitro action 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.

Dataset Information

RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

Publications

RecA-independent recombination: Dependence on the Escherichia coli RarA protein.

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