Excision of 3 ' termini by the Trex1 and TREX2 3 '-> 5 ' exonucleases - Characterization of the recombinant proteins

The excision of nucleotides from DNA 3' termini is an important step in DNA replication, repair, and recombination pathways to generate correctly base paired termini for subsequent processing. The mammalian TREX1 and TREX2 pr oteins contain potent 3'-->5' exonucleases capable of functioning in this c apacity. To study the activities of these exonucleases we have developed st rategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical chara cterization. The Trex1 and TREX2 proteins are homodimers that exhibit robus t 3' excision activities with very similar preferred reaction conditions an d preferences for specific DNA substrates. In a steady-state kinetic analys is, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2. The Michaelis constants derived from these data indicat e similar apparent k(cat) values of 22 s(-1) for Trex1 and 16 s(-1) for TRE X2 using single-stranded oligonucleotides. The apparent K, values of 19 nM for Trex1 and 190 nM for TREX2 suggest relatively high affinities for DNA f or both Trex1 and TREX2. An exonuclease competition assay was designed usin g heparin as a nonsubstrate inhibitor with a series of partial duplex DNAs to delineate the substrate structure preferences for 3' nucleotide excision by Trex1 and TREX2. The catalytic properties of the TREX proteins suggest roles for these enzymes in the 3' end-trimming processes necessary for prod ucing correctly base paired 3' termini.