Non-homologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair in mammalian cells and depends, among other things, on the DNA end-binding Ku70/80 hetero-dimer. To investigate the function of Ku in NHEJ we have compared the ability of cell-free extracts from wild-type CHO-K1 cells, Ku80-deficient xrs6 cells and Ku80-cDNA-complemented xrs6 cells (xrs6-Ku80) to rejoin different types of DSB in vitro. While the two Ku80-proficient extracts were highly efficient and accurate in rejoining all types of DNA ends, the xrs6 extract displayed strongly decreased NHEJ efficiency and accuracy. The lack of accuracy is most evident in non-homologous terminus configurations containing 3'-overhangs that abut a 5'-overhang or blunt end. While the sequences of the 3'-overhangs are mostly preserved by fill-in DNA synthesis in the Ku80-proficient extracts, they are always completely lost in the xrs6 extract so that, instead, small deletions displaying microhomology patches at their breakpoints arise. In summary, our results are consistent with previous results from Ku-deficient yeast strains and indicate that Ku may serve as an alignment factor that not only increases NHEJ efficiency but also accuracy. Furthermore, a secondary NHEJ activity is present in the absence of Ku which is error-prone and possibly assisted by base pairing interactions.

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3-overhangs and a onebase gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3 3 5 resection before ligation, was formed. Similar results were seen for a substrate with 5-overhangs and recessed 3-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation.

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.

Non-homologous end joining (NHEJ), the major pathway of double-strand break (DSB) repair in mammalian cells, comprises two subpathways: one that requires the three core factors Ku70/80, DNA-PKcs and XRCC4/LigIV (DNA-PK-dependent NHEJ) and the other that is independent of these factors. Using a cell-free NHEJ assay, we have investigated the ability of three Chinese hamster ovary (CHO) mutants deficient in Ku80 (xrs6), DNA-PKcs (XR-C1) and XRCC4 (XR-1) in comparison with CHO-K1 wild-type cells to rejoin non-compatible DSB ends. Both NHEJ efficiency and fidelity are strongly reduced in the mutants with xrs6 and XR-1 exhibiting the strongest reduction and XR-C1 displaying a phenotype intermediate between the wild-type and the other two mutants indicating a non-essential but facilitating role of DNA-PKcs in NHEJ. The decrease in fidelity in the mutants is expressed by an increase of deletion junctions formed at microhomologies (microhom) near the DSB (microhomology-mediated non-homologous end joining: microhomNHEJ). Using a novel microhomNHEJ assay, we show that microhom regions of 6-10 bp that are located directly at the DSB termini strongly enhance the mutagenic microhomNHEJ reaction even in the wild type. Due to its error proneness, DNA-PK-independent microhomNHEJ may actively promote genome instability. It will, therefore, be of increasing importance to examine NHEJ fidelity in the context with tumorigenesis and cellular senescence for which we here provide two efficient and reliable tools.