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 Each of the patients had blood withdrawn every 15 min during 12 h overnight followed by a single s.c. injection of nafarelin (1 microgram(s)/kg up to 100 microgram(s)), except two HH patients who did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.     

Resistance to chemotherapeutic antimetabolites: a function of salvage pathway involvement and cellular response to DNA damage

The inherent or acquired (induced) resistance of certain tumours to cytotoxic drug therapy is a major clinical problem. There are many categories of cytotoxic agent: the antimetabolites, e.g. methotrexate (MTX), N-phosphonacetyl-L-aspartate (PALA), 5-fluorouracil (5-FU), 6-mercaptopurine (6-TG), hydroxyurea (HU) and 1-beta-D-arabinofuranosylcytosine (AraC); the alkylating agents, e.g. the nitrogen mustards and nitrosoureas; the antibiotics, e.g. doxorubicin and mitomycin C; the plant alkaloids, e.g. vincristine and vinblastine; and miscellaneous compounds, such as cisplatin. There are also many mechanisms of drug resistance elucidated principally from in vitro studies. These include mutation of target genes, amplification of target and mutated genes, differences in repair capacity, altered drug transport and differences in nucleoside and nucleobase salvage pathways (Fox et al, 1991). The aim of the present review is to evaluate in detail the mechanisms of response of both normal and tumour cells to three chemotherapeutic antimetabolites, MTX, PALA and 5-FU, which are routinely used in the clinic either alone or in combination to treat some of the commonest solid tumours, e.g. breast, colon, gastric and head and neck. The normal and tumour cell response to these agents will be discussed in relation to the operation of the known alternative 'salvage pathways' of DNA synthesis and current theories of DNA damage response.     

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.     

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.     

On the mechanism of RecA-mediated repair of double-strand breaks: no role for four-strand DNA pairing intermediates

OBJECTIVE: To determine the effects of human seminal fluid on cervical paracellular resistance. DESIGN: Experimental study. SETTING: Healthy volunteers in an academic research environment; cultures of human CaSki cells on filters, with phenotypic characteristics of the endocervix. PATIENT(S): Healthy men donating sperm to a sperm bank. INTERVENTION(S): Seminal fluid was obtained as the discarded fluid from ejaculates. MAIN OUTCOME MEASURE(S): Changes in transepithelial electrical resistance across CaSki cells on filters were determined in an Ussing chamber from successive measurements of the short-circuit current and the transepithelial potential difference. Changes in the dilution potential (and hence in the ratio of Cl- to Na+ mobilities) were determined after lowering the NaCl concentration in the luminal solution. RESULT(S): Seminal fluid increased transepithelial electrical resistance acutely (t1/2, 2 minutes), reversibly, and in a dose-related manner (ED50, 1%). The effect of seminal fluid was abolished when the extracellular calcium level was lowered, and the increase in transepithelial electrical resistance correlated with a decrease in the ratio Cl- to Na+ mobilities, indicating an increase in the resistance of the tight junctional complex. The increase in transepithelial electrical resistance in response to seminal fluid was nonadditive to that of sn-1,2-dioctanoyl diglyceride (a stable diacylglyceride and activator of protein kinase C), and it was abolished by prolonged preincubation with the phorbol ester phorbol 12-myristate 13-acetate (to downregulate protein kinase C) or with staurosporin (to inhibit protein kinase C), suggesting that seminal fluid acts through a protein kinase C-dependent mechanism. Slower (t1/2, 3.3 minutes) increases in transepithelial electrical resistance occurred when seminal fluid was added only to the luminal or the subluminal solution. Treatment with pertussis toxin, adenosine triphosphatase, or trypsin had no effect on the changes in transepithelial electrical resistance. Seminal fluid increased cytosolic calcium, but changes in cytosolic calcium are not important for the increases in transepithelial electrical resistance, suggesting that the effect of seminal fluid is not receptor-mediated. Preliminary studies indicate that the factor(s) in seminal fluid that increases transepithelial electrical resistance is a labile, low molecular weight (< 10 kd) lipid. CONCLUSION(S): Seminal fluid may regulate cervical mucus production in vivo by modulating endocervical permeability.     

Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through Cdc25

In response to DNA damage, mammalian cells prevent cell cycle progression through the control of critical cell cycle regulators. A human gene was identified that encodes the protein Chk1, a homolog of the Schizosaccharomyces pombe Chk1 protein kinase, which is required for the DNA damage checkpoint. Human Chk1 protein was modified in response to DNA damage. In vitro Chk1 bound to and phosphorylated the dual-specificity protein phosphatases Cdc25A, Cdc25B, and Cdc25C, which control cell cycle transitions by dephosphorylating cyclin-dependent kinases. Chk1 phosphorylates Cdc25C on serine-216. As shown in an accompanying paper by Peng et al. in this issue, serine-216 phosphorylation creates a binding site for 14-3-3 protein and inhibits function of the phosphatase. These results suggest a model whereby in response to DNA damage, Chk1 phosphorylates and inhibits Cdc25C, thus preventing activation of the Cdc2-cyclin B complex and mitotic entry.     

The 52-kd protein as a target of intermolecular spreading of the immune response to components of the SS-A/Ro-SS-B/La complex

OBJECTIVE: To determine whether immunization of healthy non-autoimmune mice with 52-kd SS-A/Ro induces a secondary antibody response to other components of the 48-kd SS-B/La-60-kd SS-A/Ro RNP complex and vice versa, since anti-52-kd antibodies have been invariably linked to these antigens in patients with Sjogren's syndrome and in mothers whose children have neonatal lupus. METHODS: Female BALB/c mice were immunized with 100 microg of 6xHis recombinant human 48-kd SS-B/La, 52-kd SS-A/Ro, or 60-kd SS-A/Ro proteins, or the 6xHis polypeptide control, each purified by Ni2+ affinity chromatography. Mice subsequently received booster injections with 50 microg of the same antigen every 10-21 days. Immune responses were measured by enzyme-linked immunosorbent assay (ELISA), immunoblotting of recombinant antigens, and immunoprecipitation of 35S-methionine-labeled in vitro translation products. RESULTS: Immunization with 48-kd SS-B/La resulted in anti-48-kd SS-B/La antibodies within 45 days, followed 10 days later by a secondary response to 52-kd SS-A/Ro, as measured by ELISA. Antibody spreading to 60-kd SS-A/Ro was not detected. Immunization with 52-kd SS-A/Ro resulted in rapid high-titer anti-52-kd SS-A/Ro responses within 27 days. Spreading to 48-kd SS-B/La occurred in only 1 mouse and 60-kd SS-A/Ro was detected in a minority of the mice after prolonged antigen exposure. Immunization with 60-kd SS-A/Ro led to anti-60-kd SS-A/Ro responses within 37 days, followed 3 months later by low-titer anti-48-kd SS-B/La and anti-52-kd SS-A/Ro antibodies. All primary immune responses were confirmed by immunoblotting and immunoprecipitation. While immunoblotting of the recombinant proteins revealed reciprocal intermolecular spreading in the majority of mice, immunoprecipitation clearly demonstrated that predominant spreading was generated after immunization with 48-kd SS-B/La, which consistently resulted in antibodies to 52-kd SS-A/Ro. CONCLUSION: The murine responses observed in the present study, demonstrating reciprocal intermolecular spreading to 48-kd SS-B/La, 52-kd SS-A/Ro, and 60-kd SS-A/Ro, support the linkage of 52-kd SS-A/Ro with the other proteins, despite their as-yet-undetected association in vivo. The marked recruitment of anti-52-kd SS-A/Ro responses elicited by 48-kd SS-B/La may provide a lead to exploring the physical interaction, direct or indirect, of 52-kd SS-A/Ro with the SS-A/Ro-SS-B/La RNP particle and its presentation to the immune system. These data should facilitate the establishment of a murine model of neonatal lupus.     

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.     

Architecture of protein and DNA contacts within the TFIIIB-DNA complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.     

Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.