The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

A study of the contribution of repair processes to the formation of individual radiosensitivity in human beings at the chromosome level

Results of experimental studies that we have conducted to determine the influence of repair processes as a key factor in the formation of the individual radiosensitivity of human beings are summarized. It is shown that the repair potential is only half as high in individuals who are hypersensitive to irradiation by comparison with a control group.     

The contribution of CMP kinase to the efficiency of DNA repair

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.     

The contribution of CMP kinase to the efficiency of DNA repair

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.     

Variant of ataxia-telangiectasia with low-level radiosensitivity

Summary In the present study we examined cells from several patients clinically diagnosed as having ataxia-telangiectasia (AT), for the capacity of their cells to inhibit DNA synthesis following exposure to gamma irradiation, and for the rate of spontaneous or blcomycin-induced chromosomal aberrations. Cells from two patients showed normal inhibition of DNA synthesis and levels of induced chromosomal aberrations intermediate between normal and AT cells. These two patients had only minimal immunologic impairment. These findings appear to define one distinct subset of AT.     

Genotoxic effects of radiotherapy and chemotherapy on the circulating lymphocytes of breast cancer patients. II. Alteration of DNA repair and chromosome radiosensitivity

Lymphocytes from 43 breast cancer patients were tested for their DNA-repair ability and in vitro chromosomal radiosensitivity. Lymphocytes were collected before and after treatment with radiotherapy or chemotherapy or both, and then irradiated in vitro. The aim was to detect alterations of these 2 indicators of radiosensitivity, in relation to cancer status or medical treatment. Patients before treatment were significantly deficient in DNA-repair ability but had a normal chromosomal radiosensitivity as compared to healthy donors. When assessed after treatment, DNA-repair ability and the frequency of in vitro-induced chromosome anomalies were modified according to the type of treatment. A reduced DNA-repair ability was observed for patients after radiotherapy but not after chemotherapy. In vitro-induced dicentrics and acentrics were not modified to the same extent according to the treatment. A decreased number of acentrics (the most frequently observed alteration) was preferentially associated with a more reduced DNA-repair ability. Interindividual differences of response to in vitro irradiation tested by both assays were observed between patients who had undergone similar treatments. The possibility that these assays could be used for predicting individual susceptibility to radiation or chemotherapy drug exposure is discussed.     

Features of the radiosensitivity of cells from ataxia-telangiectasia patients]

Cell survival, single and double DNA strand breaks formation and removal, spontaneous and induced chromosome aberrations and sister chromatid exchange (SCE) levels in gamma-irradiated cells of patients with ataxia-telangiectasia (AT) were studied. Except SCE all of the above indexes of AT cells sensitivity, were higher, than in normal human cells, but lower, than it is commonly characteristic of AT cells in literature. A conclusion is that the analysed AT cells belong to the AT-variant form. Possible mechanisms of high radiosensitivity of AT cells, accompanied by radioresistance of DNA replication, are discussed. The authors suppose that the DNA repair defect in AT cells is not primary.     

Effect of DNA repair inhibitors on the induction and repair of bleomycin-induced chromosome damage.

The current status of the L5178Y/TK^+/-→TK^-/- mouse-lymphona mutagenicity assay is described. Dose-survival-mutagenic response data are shown for 43 chemicals. Mutagenicity and cytotoxicity in the presence or absence of non-induced and/or Aroclor-induced rat-liver S-9 are compared for most of these chemicals. 25 of these for which usable carcinogenicity data exist have been used to construct an approximately linear relationship between oncogenic potency in vivo and mutagenic potency in this system in vitro; linearity between these two endpoints extends over a greater than 100 000-fold range in potencies. Several carcinogens which are negative or difficult to detect in the standard Ames assay are mutagenic in this mammalian cell system. These include natulan, sodium saccharin (lot S-1022), p,p′-DDE (a metabolite of DDT), dimethylnitrosamine, diethylnitrosamine and diethylstilbestrol.     

Progressive loss of DNA sequences from terminal chromosome deficiencies in Drosophila melanogaster.

Terminal deficiencies at the tip of the X chromosome can be induced at a high frequency (0.2-0.3%) by irradiating Drosophila females carrying a homozygous mutator (mu-2) with low doses of X-rays. These terminal deficiencies are unstable, since over a period of 3 1/2 years DNA sequences were lost from their distal ends at a rate of 75 bp per generation, presumably due to the absence of a complete wild-type telomeric structure. Breakpoints of these deletions in the 5' upstream regulatory region of the yellow gene, giving rise to a mosaic cuticle pigmentation pattern typical of the y2 type, were used to define the location of tissue-specific cis-acting regulatory elements that are required for body, wing or bristle pigmentation.     

Further mapping of an ataxia-telangiectasia locus to the chromosome 11q23 region.

We recently mapped the gene for ataxia-telangiectasia group A (ATA) to chromosome 11q22-23 by linkage analysis, using the genetic markers THY1 and pYNB3.12 (D11S144). The most likely order was cent-AT-S144-THY1. The present paper describes further mapping of the AT locus by means of a panel of 10 markers that span approximately 60 cM in the 11q22-23 region centered around S144 and THY1. Location scores indicate that three contiguous subsegments within the [S144-THY1] segment, as well as three contiguous segments telomeric to THY1, are each unlikely to contain the AT locus, while the more centromeric [STMY-S144] segment is most likely to contain the AT locus. These data, together with recent refinements in the linkage and physical maps of 11q22-23, place the AT locus at 11q23.     

DNA double-strand break repair and chromosome translocations

Translocations are genetic aberrations that occur when a broken fragment of a chromosome is erroneously rejoined to another chromosome. The initial event in the creation of a translocation is the formation of a DNA double-strand break (DSB), which can be induced both under physiological situations, such as during the development of the immune system, or by exogenous DNA damaging agents. Two major repair pathways exist in cells that repair DSBs as they arise, namely homologous recombination, and non-homologous end-joining. In some situations these pathways can function inappropriately and rejoin ends incorrectly to produce genomic rearrangements, including translocations. Translocations have been implicated in cancer because of their ability to activate oncogenes. Due to selection at the level of the DNA, the cell, and the tissue certain forms of cancer are associated with specific translocations that can be used as a tool for diagnosis and prognosis of these cancers.     

The electrostatic contribution to DNA base-stacking interactions.

Base-stacking and phosphate-phosphate interactions in B-DNA are studied using the finite difference Poisson-Boltzmann equation. Interaction energies and dielectric constants are calculated and compared to the predictions of simple dielectric models. No extant simple dielectric model adequately describes phosphate-phosphate interactions. Electrostatic effects contribute negligibly to the sequence and conformational dependence of base-stacking interactions. Electrostatic base-stacking interactions can be adequately modeled using the Hingerty screening function. The repulsive and dispersive Lennard-Jones interactions dominate the dependence of the stacking interactions on roll, tilt, twist, and propellor. The Lennard-Jones stacking energy in ideal B-DNA is found to be essentially independent of sequence.     

Targeting double-strand breaks to replicating DNA identifies a subpathway of DSB repair that is defective in ataxia-telangiectasia cells.

The critical cellular defect(s) and basis for cell killing by ionizing radiation in ataxia-telangiectasia (A-T) are unknown. We use the topoisomerase I inhibitor camptothecin (CPT), which kills mainly S-phase cells and induces DSBs predominantly in replication forks, to show that A-T cells are defective in the repair of this particular subclass of DSBs. CPT-treated A-T cells reaching G2 have abnormally high levels of chromatid exchanges (viewed as prematurely condensed G2 chromosomes); aberrations in normal cells are mostly chromatid breaks. Transfectants of A-T cells with the wild-type ATM cDNA are corrected for CPT sensitivity, chromatid aberrations, and the DSB repair defect. These data suggest that in normal cells ATM, the A-T protein, probably recognizes DSBs in active replicons and targets the repair machinery to the breaks; in addition, the ATM protein is involved in the suppression of low-fidelity, adventitious rejoining between replication-associated DSBs. The loss of ATM functions therefore leads to genome destabilization, sensitivity to DSB-inducing agents and to the cancer-promoting illegitimate exchange events that follow.     

The contribution of DNA repair and antioxidants in determining cell type-specific resistance to oxidative stress

The aims of this study were; (i) to elucidate the mechanisms involved in determining cell type-specific responses to oxidative stress and (ii) to test the hypothesis that cell types which are subjected to high oxidative burdens in vivo, have greater oxidative stress resistance. Cultures of the retinal pigment epithelium (RPE), corneal fibroblasts, alveolar type II epithelium and skin epidermal cells were studied. Cellular sensitivity to H2O2 was determined by the MTT assay. Cellular antioxidant status (CuZnSOD, MnSOD, GPX, CAT) was analyzed with enzymatic assays and the susceptibility and repair capacities of nuclear and mitochondrial genomes were assessed by QPCR. Cell type-specific responses to H2O2 were observed. The RPE had the greatest resistance to oxidative stress (P>0.05; compared to all other cell types) followed by the corneal fibroblasts (P < 0.05; compared to skin and lung cells). The oxidative tolerance of the RPE coincided with greater CuZnSOD, GPX and CAT enzymatic activity (P < 0.05; compared to other cells). The RPE and corneal fibroblasts both had up-regulated nDNA repair post-treatment (P < 0.05; compared to all other cells). In summary, variations in the synergistic interplay between enzymatic antioxidants and nDNA repair have important roles in influencing cell type-specific vulnerability to oxidative stress. Furthermore, cells located in highly oxidizing microenvironments appear to have more efficient oxidative defence and repair mechanisms.     

The synergistic effect of X-rays and deficiencies in DNA repair in P-M hybrid dysgenesis in Drosophila melanogaster.

X-rays and deficiencies in DNA repair had a synergistic effect on genetic damage associated with P-element mobility in Drosophila melanogaster. These interactions, using sterility and fecundity as endpoints, were tested in dysgenic males deficient in either excision or post-replication DNA repair. Three sublines of the Harwich P strain were used for the construction of hybrid males. These sublines differ in P-induction ability based on gonadal dysgenesis sterility (GD) and snw mutability tests, in P-element insertion site pattern, and in the types of defective P-elements, such as KP elements, they possess. A lower degree of gonadal dysgenesis was correlated with the presence of KP elements. GD sterility and snw mutability were not always correlated. Dysgenic hybrids originating from the standard reference subline, Harwich(white), were much more sensitive to the post-replication repair than the excision repair defect. In contrast, sterility of hybrids derived from the weak subline was least affected by, and that of hybrids of the strongest subline was most affected by either DNA repair deficiency. The exacerbation by X-rays of the effects of DNA repair deficiencies on genetic damage indicates that both repair mechanisms are required for processing DNA lesions induced by the combined effect of P activity and ionizing radiation.     

Recovery of DNA from the Acinetobacter calcoaceticus chromosome by gap repair.

A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.     

The organization and repair of DNA in the mammalian chromosome. III. Determination of the molecular weight of a mammalian native DNA

The necessary conditions for a unique solution of the sedimentation vs DNA molecular weight equations are considered and applied to the native DNA of the L5178Y mouse leukemia cell. A brief review and critique of the literature of sedimentation anomalies is given to demonstrate that such anomalies are not present in the data reported here. It is shown that the chromosomal DNA of L5178Y cells comes in uniform packages of 1.0 (0.5–2.0) × 10¹⁰ daltons. All pieces are of an identical size which corresponds to the DNA content of about 1/13 the average chromatid. Both the size estimate and the number of such molecules/cell are confirmed by viscoelastometry. This DNA is shown to be free of radioactively demonstrable protein and/or lipid contaminants and of the same isopycnic density as T₄ DNA. Variance analysis is applied to determine the precision of all measurements and to pinpoint major sources of error. A relationship between [η] and M is derived for native DNA in 1.0M NaCl. A necessary conclusion from these data is that mammalian chromosome models requiring degrees of polynemy greater than 16-neme (in G₁) are incorrect (to the extent that the L5178Y cell is typical of mammalian cells).     

The role of chromosome unwinding proteins in the regulation of DNA repair and replication

DNA repair and replication are regulated by cellular capability to support the definite optimal level of single strand DNA binding optimal level of single strand DNA binding proteins (SSB). SSB deficiency as well as it's overproduction in the cell cause, by means of different mechanisms, the destruction of DNA macromolecules due to impairment in timing of the nucleotides hydrolysis and resynthesis reactions. This asynchronization hinders the normal processing of DNA repair and replication.