title: Allosteric ring assembly and chemo-mechanical melting by the interaction between 5'-phosphate and λ exonuclease.
abstract: Phosphates along the DNA function as chemical energy frequently used by nucleases to drive their enzymatic reactions. Exonuclease functions as a machine that converts chemical energy of the phosphodiester-chain into mechanical work. However, the roles of phosphates during exonuclease activities are unknown. We employed λ exonuclease as a model system and investigated the roles of phosphates during degradation via single-molecule fluorescence resonance energy transfer (FRET). We found that 5' phosphates, generated at each cleavage step of the reaction, chemo-mechanically facilitate the subsequent post-cleavage melting of the terminal base pairs. Degradation of DNA with a nick requires backtracking and thermal fraying at the cleavage site for re-initiation via the formation of a catalytically active complex. Unexpectedly, we discovered that a phosphate of a 5' recessed DNA acts as a hotspot for an allosteric trimeric-ring assembly without passing through the central channel. Our study provides new insight into the versatile roles of phosphates during the processive enzymatic reaction.
authors.affiliation: School of life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Jungmin
authors.initials: J
authors.lastname: Yoo
authors.affiliation: School of life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea glee@gist.ac.kr.
authors.forename: Gwangrog
authors.initials: G
authors.lastname: Lee
mesh_terms: D000495:Allosteric Site; D020029:Base Pairing; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D010710:Phosphates / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010710:Phosphates; D014764:Viral Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; C043586:exo protein, Bacteriophage lambda