Cooperative DNA binding and communication across the dimer interface in the TREX2 3' --> 5'-exonuclease.
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.
Department of Biochemistry, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA. fperrino@wfubmc.edu
Fred W
FW
Perrino
Udesh
U
de Silva
Scott
S
Harvey
Edward E
EE
Pryor
Daniel W
DW
Cole
Thomas
T
Hollis
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000235:genetics*; D005092:Exonucleases / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009696:Nucleic Acids / Q000737:chemistry; D010750:Phosphoproteins / Q000235:genetics*; D011485:Protein Binding; D013379:Substrate Specificity; D013997:Time Factors
D009696:Nucleic Acids; D010750:Phosphoproteins; D004247:DNA; D005090:Exodeoxyribonucleases; D005092:Exonucleases; C486104:TREX2 protein, human; D000409:Alanine