Rejoining of DNA double-strand breaks in vitro by single-strand annealing.

Nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in vertebrate cells. Various studies indicated the existence of at least two different NHEJ pathways; one that joins DNA ends accurately and depends on Ku, a protein heterodimer that binds to DNA ends, and one that generates deletions and is independent of Ku. While the former pathway has been characterised in some detail, only little is known about the latter error-prone. We have partially purified such an NHEJ activity from extracts of Xenopus laevis eggs. End-joined junctions formed in the most extensively purified protein fraction displayed deletions containing short patches of sequence homology at their break points, a feature characteristic of single-strand annealing (SSA). Detailed biochemical characterisation revealed the presence of DNA ligase III, DNA polymerase epsilon, FEN-1 endonuclease, and exonuclease activities of 5'-3' and 3'-5' directionality. We show that these activities are able to correctly process proposed intermediates of SSA. Interestingly, neither Ku nor the associated DNA-dependent protein kinase were detected, indicating that the mechanism can dispense with Ku. Our findings provide evidence for the existence of an error-prone NHEJ pathway that creates deletions by microhomology-driven SSA.

Mechanisms of nonhomologous DNA end-joining in frogs, mice and men.

DNA end-joining, a process related to illegitimate recombination and capable of rejoining unrelated pairs of DNA ends in the absence of sequence homology, is considered the major pathway of double-strand break (DSB) repair in mammalian cells. Whole cell and nuclear extracts from three human and one mouse cell line were investigated for their capacities to promote nonhomologous DNA end-joining and their relative activities of DNA-PK, a mammalian DNA end-binding protein complex implicated in DSB-repair. The levels of DNA end-joining and the spectra of junctions of the human systems were identical with the ones of a previously described cell-free joining system derived from Xenopus laevis eggs. Due to the presence of potent 3'-5'-exonuclease activities the mouse system displayed decreased levels of DNA end-joining and larger fractions of junctions containing deletions but otherwise the basic mechanisms of junction formation appeared to be identical with the Xenopus system. DNA-PK activity was found to be equally low in the Xenopus and the mouse system but 4- to 6-fold increased in the human systems. Our results suggest that the mechanisms of DNA end-joining may be modulated by the level of exonuclease activities and/or DNA end-protecting factors but are otherwise highly conserved in vertebrate cells.

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 approximately 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 2nt 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.

Nonhomologous DNA end joining of synthetic hairpin substrates in Xenopus laevis egg extracts.

Processes of DNA end joining are assumed to play a major role in the elimination of DNA double-strand breaks (DSB) in higher eucaryotic cells. Linear plasmid molecules terminated by nonhomologous restriction ends are the typical substrates used in the analysis of joining mechanisms. However, due to their limited structural variability, DSB ends generated by restriction cleavage cover probably only part of the total spectrum of naturally occurring DSB termini. We therefore devised novel DNA substrates consisting of synthetic hairpin-shaped oligonucleotides which permit the construction of blunt ends and 5'- or 3'-protruding single-strands (PSS) of arbitrary sequence and length. These substrates were tested in extracts of Xenopus laevis eggs known to efficiently join linear plasmids bearing nonhomologous restriction termini (Pfeiffer and Vielmetter, 1988). Sequences of hairpin junctions indicate that the short hairpins are joined by the same mechanisms as the plasmid substrates. However, the bimolecular DNA end joining reaction was only detectable when both hairpin partners had a minimal duplex stem length of 27bp and their PSS-tails did not exceed 10nt.