Abstract
Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability. The biochemical attributes of Exo1 have been extensively characterized via conventional assays. However, the key step governing its activation remains elusive. Extending the previous finding that Exo1 can digest a randomly selected single-stranded DNA (ssDNA) but not a poly(dT) oligonucleotide and using purified recombinant Exo1 and nuclease and electrophoretic mobility shift assays, here we determined that DNA hairpins with a stem size of 4 bp or longer are able to activate Exo1-mediated digestion of ssDNA. We further provide evidence suggesting that Exo1 uses an evolutionarily conserved residue, Lys185 This residue interacted with the phosphate group bridging the third and fourth nucleotide on the digestion strand of the substrate DNA for duplex recognition, critical for Exo1 activation on not only ssDNA but also dsDNA. Additionally, the defect of an exo1-K185A mutant in duplex digestion was partially rescued by longer overhanging DNA. However, we noted that the enhanced Exo1 nuclease activity by longer overhanging DNA is largely eliminated by replication protein A (RPA), likely because of the previously reported RPA activity that strips Exo1 off the ssDNA. We conclude that duplex DNA contact by Exo1 is a general mechanism that controls its activation and that this mechanism is particularly important for digestion of duplex DNA whose nascent ssDNA is bound by RPA.
Highlights
Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability
Exo1 is an evolutionarily conserved Rad2 family nuclease that plays a multifaceted role in DNA metabolic pathways governing genome stability, including mismatch repair [1,2,3], homologous recombination (HR)2–mediated double-strand break (DSB) repair [4], telomere maintenance, lagging strand maturation, and recovery of stalled replication forks [5, 6]
SsDNA digestion by Exo1 is not due to its preference to cleave at a certain nucleotide
Summary
Exonuclease 1 (Exo1) is an evolutionarily conserved eukaryotic nuclease that plays a multifaceted role in maintaining genome stability. We further provide evidence suggesting that Exo uses an evolutionarily conserved residue, Lys185 This residue interacted with the phosphate group bridging the third and fourth nucleotide on the digestion strand of the substrate DNA for duplex recognition, critical for Exo activation on ssDNA and dsDNA. Binding of hEXO1 to the duplex region of its substrate DNA was captured as the first snapshot in a further time-resolved crystallography study [22], which suggests duplex capture as a critical step in Exo1/hEXO1 activation This premise remains to be tested in functional assays. Duplex contact by Exo is a general mechanism to control Exo activation
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