E. coli Exonuclease I is a 3" to 5" exonuclease for single-stranded DNA degradation, resulting in the production of 5"-phosphate mononucleotides from the 3"-hydroxyl termini of single-stranded DNA. This 3" to 5" exonucleaseis highly specific for single-stranded DNA and does not react with double-stranded DNA or RNA. Exonuclease I is inactivated by heat treatment at 80°C for 15 minutes.