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J. Biol. Chem., Vol. 283, Issue 31, 21441-21452, August 1, 2008

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Cooperative DNA Binding and Communication across the Dimer Interface in the TREX2 3' 5'-Exonuclease^*

From the Department of Biochemistry, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157

The activity of human TREX2-catalyzed 3' 5'-deoxyribonuclease 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 K_m values for DNA substrate with no effect on k_cat 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 TREX2^WT/H188A heterodimer compared with the TREX2^WT homodimer, contrasting the 2-fold lower activity measured in the TREX2^{WT/R163A,R165A,R167A} heterodimer. The measured activity in TREX2^WT/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 turnover kinetic assays identify DNA binding as the rate-limiting step in TREX2 catalysis.

Received for publication, May 12, 2008 , and in revised form, June 3, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM069962 (to F. W. P.). This work was also supported by American Cancer Society Grant RSG-04-187-01-GMC (to T. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3 and additional references.

¹ To whom correspondence should be addressed. Tel.: 336-716-4349; Fax: 336-716-7671; E-mail: fperrino{at}wfubmc.edu.

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