A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA

Background: Double-strand breaks in chromosomal DNA of eucaryotic cells are assumed to be repaired by mechanisms of illegitimate recombination capable of direct rejoining of the broken ends. Cell-free extracts of Xenopus laevis eggs efficiently perform these end joining reactions with any pair of noncomplementary DNA termini whose single-stranded 5′- or 3′-overhangs do not exceed a length of ≈ 10 nt. Results: Using hairpin-shaped oligonucleotides that allow the construction of double-strand break termini with 5′- or 3′-overhangs of defined length and sequence we show that 5′-overhangs of more than 9–10 nt are exonucleolytically resected in the extract to produce shorter 5′-overhangs that can be metabolized in the end joining reaction. 5′-recessed ends in double-stranded DNA with 3′-overhangs of more than 2 nt as well as the 5′-ends of single-stranded DNA also serve as substrates for the exonuclease activity. In all cases, oligomers of about 10 nt are released from the 5′-ends. Conclusions: We describe here a novel 5′-exonuclease activity present in eggs from Xenopus laevis that reproducibly removes decameric oligonucleotides from 5′-ends of double- and single-stranded DNA. A possible function of this unusual activity is discussed in the context of homologous and illegitimate genetic recombination processes.