Nonhomologous recombination in human cells

Nonhomologous recombination (NHR) is a major pathway for the repair of chromosomal double-strand breaks in the DNA of somatic cells. In this study, a comparison was made between the nonhomologous end joining of transfected adenovirus DNA fragments in vivo and the ability of purified human proteins to catalyze nonhomologous end joining in vitro. Adenovirus DNA fragments were shown to be efficiently joined in human cells regardless of the structure of the ends. Sequence analysis of these junctions revealed that the two participating ends frequently lost nucleotides from the 3' strands at the site of the joint. To examine the biochemical basis of the end joining, nuclear extracts were prepared from a wide variety of mammalian cell lines and tested for their ability to join test plasmid substrates. Efficient ligation of the linear substrate DNA was observed, the in vitro products being similar to the in vivo products with respect to the loss of 3' nucleotides at the junction. Substantial purification of the end-joining activity was carried out with the human immature T-cell-line HPB-ALL. The protein preparation was found to join all types of linear DNA substrates containing heterologous ends with closely equivalent efficiencies. The in vitro system for end joining does not appear to contain any of the three known DNA ligases, on the basis of a number of criteria, and has been termed the NHR ligase. The enriched activity resides in a high-molecular-weight recombination complex that appears to include and require the human homologous pairing protein HPP-1 as well as the NHR ligase. Characterization of the product molecules of the NHR ligase reaction suggests that they are linear oligomers of the monomer substrate joined nonrandomly head-to-head and/or tail-to-tail. The joined ends of the products were found to be modified by a 3' exonuclease prior to ligation, and no circular DNA molecules were detected. These types of products are similar to those required for the breakage-fusion-bridge cycle, a major NHR pathway for chromosome double-strand break repair.     

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.     

Assessment of the antiexudative and antiproliferative activities of non-steroidal anti-inflammatory drugs in inflammatory models developed in rats by subcutaneous implantation of bacterial cell walls from the dental plaque

Previous studies have shown that linearized SV40-based shuttle vectors transfected into mammalian cells are efficiently recircularized by an error-prone end-joining pathway. To determine whether and with what specificity free radical-mediated double-strand breaks are rejoined by this pathway, a structural mimic of such a break was introduced at a specific site in an SV40-based shuttle vector, by ligating purified 3'-phosphoglycolate-terminated oligonucleotides into 3' recessed ends generated in the linearized vector. These terminally blocked linear vectors were efficiently repaired and replicated when transfected into simian CV-1 cells. Sequencing across the repair joints in progeny plasmid indicated that, for a blunt-ended vector, the most frequent mechanism of rejoining was splicing at a terminal 4-base homology; however, a significant fraction of the joints retained all bases from both ends of the break, consistent with a mechanism involving simple 3'-phosphoglycolate removal, followed by blunt-end ligation. For the analogous 3'-hydroxyl terminated break, the fraction of simple blunt-end ligations was considerably higher. For a phosphoglycolate-terminated vector with cohesive ends the most frequent repair mechanism was simple ligation of the annealed cohesive ends, presumably preceded by phosphoglycolate removal. For all these substrates, the remaining repair joints showed small or large deletions from one or both of the ends, usually with apparent annealing at short (1-4-base) homologies. The results suggest that while breaks with 3'-phosphoglycolates can be repaired, these blocked termini represent a significant barrier to DNA end-joining, and can significantly alter its specificity. The presence of cohesive ends appears to improve markedly the fidelity of rejoining for terminally blocked double-strand breaks.     

Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.     

Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination

In mammalian cells, chromosomal double-strand breaks are efficiently repaired, yet little is known about the relative contributions of homologous recombination and illegitimate recombination in the repair process. In this study, we used a loss-of-function assay to assess the repair of double-strand breaks by homologous and illegitimate recombination. We have used a hamster cell line engineered by gene targeting to contain a tandem duplication of the native adenine phosphoribosyltransferase (APRT) gene with an I-SceI recognition site in the otherwise wild-type APRT+ copy of the gene. Site-specific double-strand breaks were induced by intracellular expression of I-SceI, a rare-cutting endonuclease from the yeast Saccharomyces cerevisiae. I-SceI cleavage stimulated homologous recombination about 100-fold; however, illegitimate recombination was stimulated more than 1,000-fold. These results suggest that illegitimate recombination is an important competing pathway with homologous recombination for chromosomal double-strand break repair in mammalian cells.     

Double-strand break repair in Ku86- and XRCC4-deficient cells

The Ku86 and XRCC4 proteins perform critical but poorly understood functions in the repair of DNA double-strand breaks. Both Ku 86- and XRCC4-deficient cells exhibit profound radiosensitivity and severe defects in V(D)J recombination, including excessive deletions at recombinant junctions. Previous workers have suggested that these phenomena may reflect defects in joining of the broken DNA ends or in protection of the ends from nucleases. However, end joining in XRCC4-deficient cells has not been examined. Here we show that joining of both matched and mismatched DNA ends occurs efficiently in XRCC4-deficient cells. Furthermore, analysis of junctions shows that XRCC4 is not required to protect the ends from degradation. However, nucleotide sequence analysis of junctions derived from joining of mismatched DNA ends in XRCC4-deficient cells revealed a strong preference for a junction containing a 7 nt homology. Similar results were obtained in Ku86-deficient cells. These data suggest that in the absence of XRCC4 or Ku86, joining is assisted by base pairing interactions, supporting the hypothesis that these proteins may participate in aligning or stabilizing intermediates in end joining.     

One-sided invasion events in homologous recombination at double-strand breaks

Two classes of homologous recombination mechanism for repair of double-strand breaks (DSBs) have been described in eukaryotes so far. One is conservative and has been explained by the double-strand break repair model (Szostak et al., 1983), whereas the other one is non-conservative and has been explained by the single-strand annealing model (Lin et al., 1984). Here, we will review data supporting the existence of another homologous recombination mechanism for double-strand break repair. We will present the one-sided invasion model that we have proposed to explain this mechanism and discuss its potential implication in various homologous recombination events.     

Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions

Double strand DNA breaks in plants are primarily repaired via non-homologous end joining. However, little is known about the molecular events underlying this process. We have studied non-homologous end joining of linearized plasmid DNA with different termini configurations following transformation into tobacco cells. A variety of sequences were found at novel end junctions. Joining with no sequence alterations was rare. In most cases, deletions were found at both ends, and rejoining usually occurred at short repeats. A distinct feature of plant junctions was the presence of relatively large, up to 1.2 kb long, insertions (filler DNA), in approximately 30% of the analyzed clones. The filler DNA originated either from internal regions of the plasmid or from tobacco genomic DNA. Some insertions had a complex structure consisting of several reshuffled plasmid-related regions. These data suggest that double strand break repair in plants involves extensive end degradation, DNA synthesis following invasion of ectopic templates and multiple template switches. Such a mechanism is reminiscent of the synthesis-dependent recombination in bacteriophage T4. It can also explain the frequent 'DNA scrambling' associated with illegitimate recombination in plants.     

The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products

The bacterial transposon Tn7 translocates by a cut and paste mechanism: excision from the donor site results from double-strand breaks at each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.     

DNA ligase IV binds to XRCC4 via a motif located between rather than within its BRCT domains

The covalent rejoining of DNA ends at single-stranded or double-stranded DNA breaks is catalyzed by DNA ligases. Four DNA ligase activities (I-IV) have been identified in mammalian cells [1]. It has recently been demonstrated that DNA ligase IV interacts with and is catalytically stimulated by the XRCC4 protein [2,3], which is essential for DNA double-strand break repair and the genomic rearrangement process of V(D)J recombination [4]. Together with the finding that the yeast DNA ligase IV homologue is essential for nonhomologous DNA end joining [5-7], this has led to the hypothesis that mammalian DNA ligase IV catalyzes ligation steps in both of these processes [8]. DNA ligase IV is characterized by a unique carboxy-terminal tail comprising two BRCT (BRCA1 carboxyl terminus) domains. BRCT domains were initially identified in the breast cancer susceptibility protein BRCA1 [9], but are also found in other DNA repair proteins [10]. It has been suggested that DNA ligase IV associates with XRCC4 via its tandem BRCT domains and that this may be a general model for protein-protein interactions between DNA repair proteins [3]. We have performed a detailed deletional analysis of DNA ligase IV to define its XRCC4-binding domain and to characterize regions essential for its catalytic activity. We find that a region in the carboxy-terminal tail of DNA ligase IV located between rather than within BRCT domains is necessary and sufficient to confer binding to XRCC4. The catalytic activity of DNA ligase IV is affected by mutations within the first two-thirds of the protein including a 67 amino-acid amino-terminal region that was previously thought not to be present in human DNA ligase IV [11].     

Homologous, homeologous, and illegitimate repair of double-strand breaks during transformation of a wild-type strain and a rad52 mutant strain of Saccharomyces cerevisiae

Different modes of in vivo repair of double-strand breaks (DSBs) have been described for various organisms: the recombinational DSB repair (DSBR) mode, the single-strand annealing (SSA) mode, and end-to-end joining. To investigate these modes of DSB repair in Saccharomyces cerevisiae, we have examined the fate of in vitro linearized replicative plasmids during transformation with respect to several parameters. We found that (i) the efficiencies of both intramolecular and intermolecular linear plasmid DSB repair are homology dependent (according to the amount of DNA used during transformation [100 ng or less], recombination between similar but not identical [homeologous] P450s sequences sharing 73% identity is 2- to 18-fold lower than recombination between identical sequences); (ii) the RAD52 gene product is not essential for intramolecular recombination between homologous and homeologous direct repeats (as in the wild-type strain, recombination occurs with respect to the overall alignment of the parental sequences); (iii) in contrast, the RAD52 gene product is required for intermolecular interactions (the rare transformants which are obtained contain plasmids resulting from deletion-forming intramolecular events involving little or no sequence homology); (iv) similarly, sequencing data revealed examples of intramolecular joining within the few terminal nucleotides of the transforming DNA upon transformation with a linear plasmid with no repeat in the wild-type strain. The recombinant junctions of the rare illegitimate events obtained with S. cerevisiae are very similar to those observed in the repair of DSB in mammalian cells. Together, these and previous results suggest the existence of alternative modes for DSB repair during transformation which differ in their efficiencies and in the structure of their products. We discuss the implications of these results with respect to the existence of alternative pathways and the role of the RAD52 gene product.     

Expansions and contractions in a tandem repeat induced by double-strand break repair

Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.     

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

Calcitonin versus etidronate for the treatment of postmenopausal osteoporosis: a meta-analysis of published clinical trials

DNA double-strand breaks are very genotoxic lesions that can result in chromosome aberrations. The current view is that DNA double-strand breaks are repaired most efficiently through homologous recombination in yeast and simple end-joining in mammalian cells. However, recent experiments reveal that both repair pathways are conserved from yeast to mammals, including humans. The challenge ahead is to put the different pieces of the jigsaw together into coherent mechanisms for both pathways and to determine their relative contributions to ionizing-radiation resistance and to the prevention of genetic instability and carcinogenesis.     

Double-strand end repair via the RecBC pathway in Escherichia coli primes DNA replication

To study the relationship between homologous recombination and DNA replication in Escherichia coli, we monitored the behavior of phage lambda chromosomes, repressed or not for lambda gene activities. Recombination in our system is stimulated both by DNA replication and by experimentally introduced double-strand ends, supporting the idea that DNA replication generates occasional double-strand ends. We report that the RecBC recombinational pathway of E. coli uses double-strand ends to prime DNA synthesis, implying a circular relationship between DNA replication and recombination and suggesting that the primary role of recombination is in the repair of disintegrated replication forks arising during vegetative reproduction.     

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.     

Genetic manipulation of genomes with rare-cutting endonucleases

DNA double-strand breaks (DSBs) pose a threat to the genomic integrity of a cell. The failure to heal a break or the inappropriate repair of a break can result in the loss of genetic information and other potentially deleterious consequences, such as chromosomal translocations. Recent developments using rare-cutting endonucleases have allowed investigators to introduce one or a few DSBs into complex genomes. Such studies have begun to elucidate the complex mechanisms of nonhomologous and homologous repair used by mammalian cells to repair these lesions. A key finding is that gene targeting is stimulated two to three orders of magnitude by a DSB at the target locus. Thus, the use of rare-cutting endonucleases and the co-opting of cellular repair mechanisms might provide scientists with another tool for engineering changes into genomes.     

Activation of nuclear factor kappa B inflammatory bowel disease

Nonhomologous DNA end joining (NHEJ) is the major pathway for repairing double-strand DNA breaks. V(D)J recombination is a double-strand DNA breakage and rejoining process that relies on NHEJ for the joining steps. Here we show that the targeted disruption of both DNA ligase IV alleles in a human pre-B cell line renders the cells sensitive to ionizing radiation and ablates V(D)J recombination. This phenotype can only be reversed by complementation with DNA ligase IV but not by expression of either of the remaining two ligases, DNA ligase I or III. Hence, DNA ligase IV is the activity responsible for the ligation step in NHEJ and in V(D)J recombination.