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 ε, 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 microhomologydriven SSA.Keywords : DSB repair; nonhomologous DNA end joining; single-strand annealing; illegitimate recombination; ligation.DNA double-strand breaks (DSB), probably the most disruptive form of DNA damage, may arise spontaneously by various cellular processes [1,2] or after exposure to DNA-damaging agents, such as ionising radiation (IR) [3]. If left unrepaired, DSB may result in broken chromosomes and cell death and if repaired improperly, they can cause mutations, chromosome rearrangements and oncogenic transformation. Repair of DSB is achieved either by homologous recombination which requires an intact copy of the broken DNA sequence provided by the sister chromatid [4], or by nonhomologous DNA end joining (NHEJ) which rejoins the broken ends directly and appears to be the main pathway in vertebrates [5,6]. Mammalian cells defective in DSB repair fall into four complementation groups [7]. The corresponding genes (XRCC4-7) encode the XRCC4 protein that associates with and stimulates DNA ligase IV [8,9], and the three components of the DNA-dependent protein kinase (DNA-PK) represented by the 86-kDa and 70-kDa subunits of the DNA end-binding Ku heterodimer and the catalytic subunit of the protein kinase (DNA-PK CS ), respectively [5,10].Studies in rad52 yeast strains in which the yeast Ku70/86 and/or DNA ligase IV homologues were knocked out showed that efficient NHEJ requires Ku and DNA ligase IV. In the absence of these proteins, a second less efficient NHEJ pathway is still functional which creates deletions [11,12]. Consistent with this, increased frequencies of imprecise end joining were also observed in the xrs6 hamster cell line which is deficient in Ku86 [13]. Together with the results from partially puri...

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.

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.

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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 singlestranded 5