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 proteins contain potent 3'-->5' exonucleases capable of functioning in this capacity. To study the activities of these exonucleases we have developed strategies to express and purify the recombinant mouse Trex1 and human TREX2 proteins in Escherichia coli in quantities sufficient for biochemical characterization. The Trex1 and TREX2 proteins are homodimers that exhibit robust 3' excision activities with very similar preferred reaction conditions and preferences for specific DNA substrates. In a steady-state kinetic analysis, oligonucleotide substrates were used to measure 3' nucleotide excision by Trex1 and TREX2. The Michaelis constants derived from these data indicate similar apparent kcat values of 22 s(-1) for Trex1 and 16 s(-1) for TREX2 using single-stranded oligonucleotides. The apparent KM values of 19 nm for Trex1 and 190 nm for TREX2 suggest relatively high affinities for DNA for both Trex1 and TREX2. An exonuclease competition assay was designed using 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 producing correctly base paired 3' termini.
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
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Mazur
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Perrino
D000818:Animals; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008322:Mammals; D017895:Manganese Compounds / Q000494:pharmacology; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D002712:Chlorides; D004277:DNA, Single-Stranded; D017895:Manganese Compounds; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C486104:TREX2 protein, human; C429526:three prime repair exonuclease 1; C025340:manganese chloride