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Unleashing the True Recombination Power of RecA by Conjugational Screening of Random Mutants in the MAW Region
Author(s) -
Herlambang Karina,
Kim TaeJin,
Cox Michael
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.878.6
Subject(s) - homologous recombination , recombination , plasmid , flp frt recombination , mutant , biology , site specific recombination , genetics , dna , gene , escherichia coli , cre lox recombination , dna repair , genetic recombination , recombinase , transgene , genetically modified mouse
The Escherichia coli RecA protein plays a major role in DNA repair through homologous recombination. However, recombination activity can have deleterious consequences and its dependency on cofactors suggest that the RecA protein is not optimized for recombination function. One region of RecA that is relatively unexplored, despite being the most conserved part, is the ‘Makes ATP Work' (MAW) motif. The MAW motif is proposed to modulate interactions with DNA and acts as a conformation switch that responds to different cofactors. Therefore, it is desirable to create a cofactor‐independent RecA protein with enhanced recombination functions. The work presented here is a novel approach using conjugational screening developed in the lab to find E.coli variants in MAW region with enhanced recombination activities. The initial mutated recA plasmid libraries are generated using a modification of the QuickChange™ method, in which two highly mutagenic oligonucleotides are used simultaneously in two successive phases of DNA synthesis where the product from the first phase is used to prime the remainder of the plasmid. The selection of active variants with enhanced recombination activities is performed by exploiting RecA‐mediated recombination during bacterial conjugation into recA‐ HFR recipient cells. Using antibiotic and growth selection genes, cells will be screened for variants capable of performing six genomic crossovers to identify the most prominent mutations. The study aims to unravel the mechanism of the MAW motif in recombination by characterizing the biochemical features of selected mutants. The newly discovered RecA mutants will expand the potential applications of RecA in gene targeting and forensics applications. NIH‐GM32335