Cooperative DNA binding and communication across the dimer interface in the TREX2 3′ → 5′-exonuclease

Abstract

The activity of human TREX2-catalyzed 3′ → 5′-deoxyribo- nuclease has been analyzed in steady-state and single turnover kinetic assays and in equilibrium DNA binding studies. These kinetic data provide evidence for cooperative DNA binding within TREX2 and for coordinated catalysis between the TREX2 active sites supporting a model for communication between the protomers of a TREX2 dimer. Mobile loops positioned adjacent to the active sites provide the major DNA binding contribution and facilitate subsequent binding into the active sites. Mutations of three arginine residues on these loops cause decreased TREX2 activities by up to 60-fold. Steady-state kinetic assays of these arginine to alanine TREX2 variants result in increased Km values for DNA substrate with no effect on kcat values indicating contributions exclusively to DNA binding by all three of the loop arginines. TREX2 heterodimers were prepared to determine whether exonuclease activity in one protomer is communicated to the opposing protomer. Evidence for communication across the dimer interface is provided by the 7-fold lower catalytic activity measured in the TREX2WT/H188A heterodimer compared with the TREX2WT homodimer, contrasting the 2-fold lower activity measured in the TREX2WT/R163A,R165A,R167A heterodimer. The measured activity in TREX2WT/H188A heterodimer indicates that defective catalysis in one protomer reduces activity in the opposing protomer. A DNA binding analysis of TREX2 and the heterodimers indicates a cooperative binding effect within the TREX2 protomer. Finally, single turn-over kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.