Identification and expression of the TREX1 and TREX2 cDNA sequences encoding mammalian 3 '-> 5 ' exonucleases

The 3'-->5' exonucleases catalyze the excision of nucleoside monophosphates from the 3' termini of DNA. We have identified the cDNA sequences encoding two 3'-->5' exonucleases (TREX1 and TREX2) from mammalian cells. The TREX1 and TREX2 proteins are 304 and 236 amino acids in length, respectively. An alysis of the TREX1 and TREX2 sequences identifies three conserved motifs t hat likely generate the exonuclease active site in these enzymes. The speci fic amino acids in these three conserved motifs suggest that these mammalia n exonucleases are most closely related to the proofreading exonucleases of the bacterial replicative DNA polymerases and the RNase T enzymes. Express ion of TREX1 and TREX2 in Escherichia coli demonstrates that these recombin ant proteins are active 3'-->5' exonucleases, The recombinant TREX1 protein was purified, and exonuclease activity was measured using single-stranded, partial duplex, and mispaired oligonucleotide DNA substrates. The greatest activity of the TREX1 protein was detected using a partial duplex DNA cont aining five mispaired nucleotides at the 3' terminus. No activity was detec ted using single-stranded RNA or an RNA-DNA partial duplex. Identification of the TREX1 and TREX2 cDNA sequences provides the genetic tools to investi gate the physiological roles of these exonucleases in mammalian DNA replica tion, repair, and recombination pathways.