Biochemical Characterization of RecBCD Enzyme from An AntarcticPseudomonasSpecies and Identification of Its Cognate Chi (χ) Sequence

Author:

Pavankumar Theetha L.ORCID,Sinha Anurag Kumar,Ray Malay K.

Abstract

ABSTRACTPseudomonas syringaeLz4W RecBCD enzyme, RecBCDPs, is a trimeric protein complex comprised of RecC, RecB, and RecD subunits. RecBCD enzyme is essential forP. syringaegrowth at low temperature, and it protects cells from low temperature induced replication arrest. In this study, we show that the RecBCDPsenzyme displays distinct biochemical behaviors. UnlikeE. coliRecBCD enzyme, the RecD subunit is indispensable for RecBCDPsfunction. The RecD motor activity is essential for the Chi-like fragments production inP. syringae, highlighting a distinct role forP. syringaeRecD subunit in DNA repair and recombination process. Further, the ssDNA-dependent endonuclease activity is notably absent in RecBCDPsenzyme. Here, we demonstrate that the RecBCDPsenzyme recognizes a unique octameric DNA sequence, 5′-GCTGGCGC-3′ (ChiPs) that attenuates nuclease activity of the enzyme when it enters dsDNA from the 3′-end. We propose that the reduced translocation activities manifested by motor-defective mutants cause cold sensitivity inP. syrinage; emphasizing the importance of DNA processing and recombination functions in rescuing low temperature induced replication fork arrest.Abbreviations:ATPAdenosine triphosphateDSBdouble-strand break‘ChiCrossover hotspot instigatorNi-NTANitrio tri-acetic acidTLCthin layer chromatographyMMCmitomycin CUV lightUltra violetABMAntarctic bacterial mediumLBLuria-Bertani medium

Publisher

Cold Spring Harbor Laboratory

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