title: Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination.
abstract: The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Taejin
authors.initials: T
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Sindhu
authors.initials: S
authors.lastname: Chitteni-Pattu
authors.affiliation: Department of Medical Physics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Cox
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Wood
authors.affiliation: Department of Microbiology, University of Massachusetts-Amherst, Amherst, Massachusetts, United States of America.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Sandler
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Cox
mesh_terms: D000595:Amino Acid Sequence; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D042822:Genomic Instability; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic
chemical_list: D029968:Escherichia coli Proteins; D011938:Rec A Recombinases