Biochemical characterization of RecBCD enzyme from an Antarctic Pseudomonas species and identification of its cognate Chi (χ) sequence

Abstract

Pseudomonas syringae Lz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential for P. syringae growth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPs enzyme displays distinct biochemical behaviors. Unlike E. coli RecBCD enzyme, the RecD subunit is indispensable for RecBCDPs function. The RecD motor activity is essential for the Chi-like fragments production in P. syringae, highlighting a distinct role for P. syringae RecD subunit in DNA repair and recombination process. Here, we demonstrate that the RecBCDPs enzyme recognizes a unique octameric DNA sequence, 50-GCTGGCGC-30 (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 30-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity in P. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.