Genetic analysis of double-strand break repair in Escherichia coli

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.     

Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways

Ku, a heterodimer of polypeptides of approximately 70 kDa and 80 kDa (Ku70 and Ku80, respectively), binds avidly to DNA double-strand breaks (DSBs). Mammalian cells defective in Ku are hypersensitive to ionizing radiation due to a deficiency in DSB repair. Here, we show that the simple inactivation of the Saccharomyces cerevisiae Ku70 homologue (Yku70p), does not lead to increased radiosensitivity. However, yku70 mutations enhance the radiosensitivity of rad52 strains, which are deficient in homologous recombination. Through establishing a rapid and reproducible in vivo plasmid rejoining assay, we show that Yku70p plays a crucial role in the repair of DSBs bearing cohesive termini. Whereas this damage is repaired accurately in YKU70 backgrounds, in yku70 mutant strains terminal deletions of up to several hundred bp occur before ligation ensues. Interestingly, this error-prone DNA repair pathway utilizes short homologies between the two recombining molecules and is thus highly reminiscent of a predominant form of DSB repair that operates in vertebrates. These data therefore provide evidence for two distinct and evolutionarily conserved illegitimate recombination pathways. One of these is accurate and Yku70p-dependent, whereas the other is error-prone and Yku70-independent. Furthermore, our studies suggest that Yku70 promotes genomic stability both by promoting accurate DNA repair and by serving as a barrier to error-prone repair processes.     

Gene conversion tracts from double-strand break repair in mammalian cells

A new assay termed the dome disappearance method for classical swine fever virus (CSFV) using FS-L3 cells with serum-free culture medium was developed. The CSFV live vaccine GPE- strain grows well and shows a slight cytopathic effect (CPE) in FS-L3 cells. This CPE results in the disappearance of the unique fluid-filled multicellular domes on a single monolayer of FS-L3 cells. By using this phenomenon, dome disappearance, as a marker of infection, it was possible to determine the titers of CSFV and its neutralizing antibody. The virus titer determined by this method shows a good correlation with that determined by immunochemical and interference methods. Furthermore, the amount of neutralizing antibody measured by this method also correlated with that measured by the Exaltation of Newcastle Disease Virus (END) neutralizing method. The dome disappearance method developed in this experiment is a simple and safe procedure and has the great advantage that bovine serum, which may contain antibody against bovine viral diarrhea virus, is not necessary for the cultivation of FS-L3 cells.     

Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair

To determine the usefulness of a GnRH agonist analog as a diagnostic test to distinguish between constitutional delay of growth (CGD) in boys with Tanner stage I of sexual development and patients with hypogonadotropic hypogonadism (HH), we evaluated six boys (mean age 15 yr 4 m) and five HH patients (mean age 20 yr 4 m). In addition, 20 normal healthy men aged 21 yr to 50 yr received either nafarelin or GnRH followed two weeks later by the other test in order to compare the efficacy of each of these tests and to evaluate the optimal sampling times for the nafarelin test. All subjects were healthy, and had not received hormonal replacement for at least 2 months prior to enrollment in the study. Each man had four baseline blood samples before and at timed intervals following the administration of either GnRH or nafarelin. Each of the patients had blood withdrawn every 15 min during 12 h overnight followed by a single s.c. injection of nafarelin (1 microgram(s)/kg up to 100 microgram(s)), except two HH patients who did not have an overnight study. Blood samples were obtained at timed intervals for 24 h. LH, FSH, T and E2 were measured by RIA. Baseline concentrations of plasma LH, FSH and T were similar before the administration of either GnRH or nafarelin in the group of normal men. Peak stimulation of plasma LH, FSH and T released by nafarelin was significantly higher, and it took a longer time to reach the peak maximum, than after GnRH (p < 0.001). Mean nocturnal LH was 5.5 +/- 0.9 IU/I for the CGD group, and 2.7 +/- 0.7 IU/I for HH (p < 0.02). Mean nocturnal FSH was 5.1 +/- 1.0 and 2.5 +/- 0.2 IU/I whereas mean nocturnal T concentrations were 4.2 +/- 0.8 and 0.7 +/- 0.2 nmol/I (CGD vs HH, respectively, p < 0.02). Peak LH responses to nafarelin were 36.9 +/- 8.9 IU/I for the CGD group, and 7.0 +/- 2.0 IU/I for the HH group (p < 0.001). Peak FSH released by nafarelin was 14.2 +/- 2.4 IU/I for the CGD group and 4.8 +/- 2.0 IU/I for the HH group (p < 0.02). Peak T was reached 24 h following nafarelin injection and was 5.7 +/- 1.7 nmol/I for the CGD group and 0.3 +/- 0.2 nmol/I for the HH group (p < 0.001). The results obtained indicate that in early stages of puberty (before detectable changes of sexual maturation) the nafarelin test, with measurements of LH, FSH and T in blood or in urine, is superior to and more practical than overnight hormonal estimates to clearly distinguish CGD from HH.     

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.     

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.     

Double-strand break repair by Ku70 requires heterodimerization with Ku80 and DNA binding functions

Heterodimers of the 70 and 80 kDa Ku autoantigens (Ku70 and Ku80) activate the DNA-dependent protein kinase (DNA-PK). Mutations in any of the three subunits of this protein kinase (Ku70, Ku80 and DNA-PKcs) lead to sensitivity to ionizing radiation (IR) and to DNA double-strand breaks, and V(D)J recombination product formation defects. Here we show that the IR repair, DNA end binding and DNA-PK defects in Ku70-/- embryonic stem cells can be counteracted by introducing epitope-tagged wild-type Ku70 cDNA. Truncations and chimeras of Ku70 were used to identify the regions necessary for DNA end binding and IR repair. Site-specific mutational analysis revealed a core region of Ku70 responsible for DNA end binding and heterodimerization. The propensity for Ku70 to associate with Ku80 and to bind DNA correlates with the ability to activate DNA-PK, although two mutants showed that the roles of Ku70 in DNA-PK activation and IR repair are separate. Mutation of DNA-PK autophosphorylation sites and other structural motifs in Ku70 showed that these sites are not necessary for IR repair in vivo. These studies reveal Ku70 features required for double-strand break repair.     

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.     

An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae

An allele of RFA1, the largest subunit of the single-stranded DNA-binding complex RP-A, was identified as a suppressor of decreased direct-repeat recombination in rad1 rad52 double mutants. In this study, we used two LEU2 direct-repeat assays to investigate the mechanism by which the rfa1-D228Y allele increases recombination. We found that both intrachromatid and sister chromatid recombination are stimulated in rfa1-D228Y strains. In a rad1 rad52 background, however, the majority of the increased recombination is caused by stimulation of deletion events by an intrachromatid recombination mechanism that is likely to be single-strand annealing. Studies in which an HO endonuclease cut was introduced between the two leu2 copies indicate that the rfa1-D228Y mutation partially suppresses the rad52 defect in recovering recombination products. Furthermore, molecular analysis of processing and product formation kinetics reveals that, in a rad52 background, the rfa1-D228Y mutation results in increased levels of recombinant products and the disappearance of large single-stranded intermediates characteristic of rad52 strains. On the basis of these results, we propose that in the absence of wild-type Rad52, the interaction of RP-A with single-stranded DNA inhibits strand annealing, and that this inhibition is overcome by the rfa1-D228Y mutation.     

DNA repair functions in heterologous cells

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.     

Gene targeting of a plasmid-borne sequence to a double-strand DNA break in Drosophila melanogaster

We report an efficient and specific gene targeting method for transforming the germ line of Drosophila melanogaster. The targeting occurs during the repair of a double-strand DNA break that is induced at the white locus by the excision of a P transposable element. The break is repaired when homologous sequence is copied from a plasmid injected into the Drosophila embryo. The procedure efficiently integrates DNA into the targeted locus of the Drosophila genome. Heterologous sequence of up to 13 kbp in length can be inserted, permitting the intergration of entire genes into a common genomic site for further study.     

3'-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends

In order to assess the possible role of human apurinic/apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3'-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5'-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3'-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3'-phosphoglycolates at a 19 base-recessed 3'-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3'-termini. There was no detectable activity of Ape toward 3'-phosphoglycolates on 1 or 2 base protruding single-stranded 3'-overhangs. The results suggest that both a single-base internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3'-termini, particularly those on 3'-overhangs.     

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.     

The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions.     

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells

Recently the concept of dissociative identity disorder (formerly known as multiple personality disorder) has attracted increasing public and scientific interest. However, it is rarely diagnosed in the clinical setting. the reported case of a 47-year-old woman with a history of child abuse demonstrates the problems of differential diagnosis. A number of psychopathologic symptoms pointed to a multiple personality disorder, but in the follow-up psychotic symptoms such as delusions, possible hallucinations and bizarre behavior clearly emerged. The differential diagnosis of dissociative identity disorder includes paranoid schizophrenia, as in the case described, borderline personality disorder, hysteria, simulation and the false memory syndrome. Finally, social and cultural factors have to be considered.     

Higher-order chromatin structure-dependent repair of DNA double-strand breaks: modeling the elution of DNA from nucleoids

A 36-year-old male with unspecific symptoms and normal physical examination had right cardiac enlargement on chest X-ray. Two-dimensional echocardiographic and thoracic computed tomography demonstrated an intracardiac mass. The tumor was surgically resected and the pathological diagnosis was mixed-type epicardial hemangioma. We discuss this case and review the literature.     

Induction and repair of double-strand breaks in the replicating DNA of HeLa cells

The photochemical (lambda < 400 nm) decomposition of some monocyclic and polycyclic nitramines produces .NO2, which can be detected in the respective nitramine crystals at 77 K by EPR (electron paramagnetic resonance). In solutions of perdeutero-dimethylsulfoxide (DMSO-d6) the .NO2 produced by photolytic decomposition of dissolved nitramines can be spintrapped by the solvent to give a radical having the structure CD3-(SO2)-(NO.)-CD3. In this article, we examine this reaction for two nitramines: cyclotrimethylenetrinitramine (RDX) and hexanitrohexaazaisowurzitane (HNIW), which are energetic materials. The decay of the spin-adduct radical (I) follows first-order kinetics for both nitramines studied, having a rate constant (k) of congruent to 7.1 x 10(-4) s-1. The net growth in spin concentration of (1) measured from EPR spectra is fitted by a first-order rate equation taking into account the simultaneous competitive decay rate of spin adduct (I). Using the rate data and EPR spin concentration data, the ratio of free .NO2 produced per parent nitramine molecule is estimated as 1:1 for RDX and 4:1 for HNIW. Biological implications of trapping of .NO2 by dimethyl sulfoxide are discussed.