Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences

Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM^−/− cells. In addition, BLM^−/− cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM^−/− cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.The RecQ helicases, a subfamily of DNA helicases, carry out the unwinding of duplex DNA in the 3′ to 5′ direction. Homologs of RecQ have been identified in a wide range of organisms, from budding yeast to humans (reviewed in Ref. 1). There are five human RecQ family proteins: Blm, Wrn, RecQ1, RecQ4, and RecQ5. The BLM, WRN, and RECQ4 genes are mutated in Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively (1–3). A hallmark of Bloom syndrome cells is the drastic increase in the level of sister chromatid exchange (SCE),⁴ which results from homologous recombination (HR) associated with cross-over of the DNA damage caused during DNA replication (4, 5). It is therefore believed that Blm acts as an anti-recombination factor and inhibits aberrant recombination. This idea is supported by the observation that Sgs1, the yeast ortholog of Blm, facilitates the resolution of aberrant joint molecules during meiotic HR (6, 7) and following replication blockage (8).HR plays a critical role in the maintenance of genome stability by repairing DNA double-strand breaks (DSBs) and releasing replication blockages at damaged template strands (9, 10). The current model for HR-mediated DSB repair is that DSBs are processed to produce a 3′ single-stranded overhang, along which Rad51 is polymerized (11, 12). The resulting Rad51-DNA filament undergoes homology search and strand invasion into intact homologous duplex DNA, leading to the formation of the D-loop structure. DNA synthesis from the invading strand followed by dissociation from the homologous duplex DNA and subsequent re-annealing of the newly synthesized strand with the other end of the DSB completes the repair. This type of HR, referred to as synthesis-dependent strand anneal (SDSA), results in sequence transfer from the intact template sequence (donor) to the damaged DNA (recipient), and accounts for the majority of mitotic HR (11, 13). Extensive strand exchange of the D-loop, on the other hand, leads to the generation of Holliday junction (HJ) intermediates. SDSA does not cause cross-overs, whereas HR involving the Holliday junction often causes cross-overs, such as SCE and meiotic HR. An increase in the level of SCE in Bloom syndrome cells therefore supports the idea that Blm suppresses the formation of HJ as well as recombinogenic DNA lesions. This idea is supported by the biochemical evidence of the Blm-dependent resolution of Holliday junctions (14). On the other hand, in Drosophila, DmBlm is known to facilitate the repair of DSB by promoting SDSA (15, 16). However, the role of Blm in SDSA in the other higher eukaryotic cells has not been defined.BLM^−/− cells established from the chicken DT40 B lymphocyte line exhibit a marked increase in the frequency of both SCE and targeted integration (17–19), as do human Bloom syndrome cells (20, 21). In this study, using the chicken DT40 cells, we investigated the role of Blm in SDSA induced by defined DNA damage. To this end, we evaluated this type of SDSA using two phenotypic assays designed to analyze Ig gene conversion and DSB-induced gene targeting. Ig gene conversion diversifies the Ig variable (V) gene through HR during in vitro passage. This reaction is initiated by activation-induced cytidine deaminase-mediated uracil formation at the functional rearranged V-region (22–24). Uracil is converted to an abasic site, probably leading to a single-strand gap (25). This lesion in the functional rearranged VJ_λ stimulates the nonreciprocal sequence transfer of a single nucleotide to several hundred nucleotides, from an array of “pseudo-V_λ” regions (donor), located upstream from the functional rearranged VJ_λ, to the rearranged V region (recipient) (26–28) (see Fig. 1A). Because donor and recipient segments have an ∼10% sequence divergence, sequential Ig gene conversion events are able to substantially diversify Ig V segments. Ig gene conversion is raised only by SDSA without the formation of a Holliday junction. Hence, phenotypic analysis of Ig gene conversion provides a unique opportunity to selectively examine the role of Blm in activation-induced cytidine deaminase-induced SDSA. Moreover, nucleotide sequence analysis of Ig gene conversion products can evaluate the accuracy of HR. Like Ig gene conversion, DSB-induced gene targeting is mediated only by SDSA. The induction of DSBs by a rare-cutting endonuclease, I-SceI, at the endogenous locus, increases the frequency of gene targeting by 3 orders of magnitudes, and the frequency of gene targeting can be evaluated by measuring the reconstitution of a marker gene (29) (see Fig. 1B).Open in a separate windowFIGURE 1.Schematic diagram of assay systems used in this study. A, principle of the Ig gene conversion assay. The predominantly sIgM-negative DT40 clone contains a frameshift in its rearranged V-J_λ segments, which can be repaired by pseudogene-templated conversion events. The rate of Ig gene conversion can be measured in subclones by flow cytometric analysis of sIgM staining. B, phenotypic assays of Ig gene conversion and DSB-induced gene targeting. Pseudo-V genes and the targeting fragment act as donors for the rearranged V_λ segment and S2neo, respectively.We here show that the loss of Blm drastically reduces the rate of Ig gene conversion without compromising its accuracy or affecting the length of the gene conversion tracts, indicating that Blm plays a role in the promotion of SDSA. This is an unexpected result, because Blm is in fact believed to suppress general HR reactions, particularly recombination between diverged homologous sequences. To understand the function of Blm in SDSA, we analyzed the effect of heterologous sequences near a DSB site on HR-dependent DSB repair. The data demonstrate that Blm can promote SDSA when there is sequence divergence between the damaged recipient DNA and the homologous donor sequence. Thus, Blm has both positive and negative effects on HR, depending upon the type of DNA damage and the step of the HR reaction.     

Effects of zinc deficiency on rice growth and genetic factors contributing to tolerance

Zinc (Zn) deficiency is the most widespread micronutrient disorder in rice (Oryza sativa), but efforts to develop cultivars with improved tolerance have been hampered by insufficient understanding of genetic factors contributing to tolerance. The objective of this paper was to examine alternative evaluation methods and to identify the most informative traits that would provide realistic information for rice breeders and to map quantitative trait loci (QTLs) associated with tolerance. Screening experiments in low-Zn nutrient solution and in a Zn-deficient field did not produce similar tolerance rankings in a set of segregating lines, which suggested that rhizosphere effects were of greater importance for lowland rice than internal Zn efficiency. The most severe symptom in the field was high plant mortality. The occurrence of leaf bronzing, usually regarded as indicative of susceptibility, did not necessarily concur with high plant mortality, which implied that both were under independent genetic control. The QTL mapping experiment conducted in the field with a population derived from a cross of IR74 (intolerant) with Jalmagna (tolerant) largely confirmed this. Four QTLs associated with plant mortality were detected, and only one of those colocalized with one of the four QTLs detected for leaf bronzing. The two most influential QTLs for plant mortality were detected on chromosomes 2 and 12. They explained 16.6% and 24.2% of the variation, and alleles of the tolerant donor parent Jalmagna reduced mortality by 16.6% and 14.8%, respectively. QTLs for plant mortality acted in a purely additive manner, whereas digenic epistatic interactions were important for leaf bronzing.     

Epithelial cells secrete interleukin-8 in response to adhesion and invasion of diffusely adhering Escherichia coli lacking Afa/Dr genes

Escherichia coli that sparsely adhere to human epithelial cells are known as diffusely adherent E. coli (DAEC), and the role of the Afa/Dr family of adhesins is now understood. Strains that do not possess Afa/Dr, however, comprise another group of DAEC, of which the pathogenicity remains unknown. The ability to induce interleukin-8 (IL-8) secretion from intestinal epithelial cells might be a feature of enterovirulent bacteria. We previously found that some Afa/Dr DAEC strains induce IL-8 by stimulating epithelial cells with flagella. The present study examines whether non-Afa/Dr DAEC can induce IL-8 in epithelial cells (HEp-2, INT407, and T84). Among 21 strains, 11 (52%; 11/21) induced as much IL-8 as high inducer strains of Afa/Dr DAEC. Adhesion did not significantly differ between high and low inducers; therefore diffuse adhesion alone is probably insufficient to induce IL-8. It was shown that IL-8 induction and the number of intracellular bacteria directly correlated. Wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase, reduced both intracellular bacteria and IL-8 secretion. Motile strains were significantly more prevalent among high (10/11) than low (4/10) inducers. However, 4 low invasive strains hardly induced IL-8 despite their motility. In conclusion, some non-Afa/Dr DAEC invoke the induction of high levels of inflammatory cytokines. Unlike Afa/Dr DAEC, however, non-Afa/Dr strains may require invasion to cause strong induction. These non-Afa/Dr high inducers can be enteropathogenic for the cytokine-inducing properties.     

Sex determination using the scapula in medieval skeletons from East Anatolia

Sex determination from skeletal human remains by discriminant function analysis is one of the methods utilized in the forensic and osteoarcheological sciences. The purpose of the present study is to establish metric standards for sex determination for medieval Anatolian populations using scapular measurements. The database for this research consisted of 93 adult skeletal remains (47 males and 46 females) from the Dilkaya medieval collection. Four measurements were taken: maximum scapular height, maximum scapular breadth, glenoid cavity height, glenoid cavity breadth, and subjected to discriminant function analysis. All measurements demonstrated some degree of sexual dimorphism, with the highest accuracy of sex determination (94.8%) obtained using maximum scapular breadth. Overall accuracies of the functions ranged from 82.9% to 95.0%, with a higher accuracy rate obtained for female skeletons than for males. Population specific discriminant formulas were developed using combinations of measurements, which can be used in ancient Anatolian populations.     

Influence of subacute treatment of some plant growth regulators on serum marker enzymes and erythrocyte and tissue antioxidant defense and lipid peroxidation in rats

This study aims to investigate the effects of the plant growth regulators (PGRs) (2,3,5-triiodobenzoic acid (TIBA), Naphthaleneacetic acid (NAA), and 2,4-dichlorofenoxyacetic acid (2,4-D)) on serum marker enzymes (aspartate aminotransferase (AST), alanin aminotransferase (ALT), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH)), antioxidant defense systems (reduced glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase (GST), and catalase (CAT)), and lipid peroxidation content (malondialdehyde = MDA) in various tissues of rats. 50 and 100 ppm of PGRs as drinking water were administered orally to rats (Sprague-Dawley albino) ad libitum for 25 days continuously. The PGRs treatment caused different effects on the serum marker enzymes, antioxidant defense systems, and the MDA content in experimented rats compared to controls. Results showed that TIBA caused a significant decrease in serum AST activity with both the dosage whereas serum CPK was significantly increased with 100 ppm dosage of TIBA. Meanwhile, serum AST, CPK, and LDH activities were significantly increased with both dosage of NAA and 2,4-D. The lipid peroxidation end-product MDA significantly increased in the all tissues treated with both dosages of PGRs without any change in the brain and erythrocyte of rats treated with both the dosages of 2,4-D. The GSH depletion in the kidney and brain tissues of rats treated with both dosages of PGRs was found to be significant. Furthermore, the GSH depletion in the erythrocyte of rats treated with both dosages of PGRs except 50 ppm dosage of 2,4-D was significant too. Also, the GSH level in the liver was significantly depleted with 50 ppm of 2,4-D and NAA, whereas the GSH depletion in the same tissue did not significantly change with the treatment. The activity of antioxidant enzymes was also seriously affected by PGRs; SOD significantly decreased in the liver, heart, kidney, and brain of rats treated with both dosages of NAA, whereas the SOD activity in the erythrocytes, liver, and heart was either significantly decreased or not changed with two doses of 2,4-D and TIBA. Although the CAT activity significantly increased in the erythrocyte and brain of rats treated with both doses of PGRs, it was not changed in the liver, heart, and kidney. Meanwhile, the ancillary enzyme GR activity significantly increased in the brain, heart, and liver but decreased in the erythrocyte and kidney of rats treated with both doses of PGRs. The drug-metabolizing enzyme GST activity significantly increased in the heart and kidney but decreased in the brain and erythrocytes of rats treated with both dosages of PGRs. As a conclusion, the results indicate that PGRs might affect antioxidant potential enzymes, the activity of hepatic damage enzymes, and lipid peroxidation dose independently. Also, the rats resisted to oxidative stress via antioxidant mechanism but the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat's tissues. These data, along with the determined changes, suggest that PGRs produced substantial systemic organ toxicity in the erythrocyte, liver, brain, heart, and kidney during the period of a 25-day subacute exposure.     

BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair

Polycomb group (PcG) proteins are major determinants of cell identity, stem cell pluripotency, and epigenetic gene silencing during development. The polycomb repressive complex 1, which contains BMI1, RING1, and RING2, functions as an E3-ubuiquitin ligase. We found that BMI1 and RING2 are recruited to sites of DNA double-strand breaks (DSBs) where they contribute to the ubiquitylation of γ-H2AX. In the absence of BMI1, several proteins dependent on ubiquitin signaling, including 53BP1, BRCA1, and RAP80, are impaired in recruitment to DSBs. Loss of BMI1 sensitizes cells to ionizing radiation to the same extent as loss of RNF8. The simultaneous depletion of both proteins revealed an additive increase in radiation sensitivity. These data uncover an unexpected link between the polycomb and the DNA damage response pathways, and suggest a novel function for BMI1 in maintaining genomic stability.     

Self-association of Calcium-binding Protein S100A4 and Metastasis

Elevated levels of the calcium-binding protein S100A4 promote metastasis and in carcinoma cells are associated with reduced survival of cancer patients. S100A4 interacts with target proteins that affect a number of activities associated with metastatic cells. However, it is not known how many of these interactions are required for S100A4-promoted metastasis, thus hampering the design of specific inhibitors of S100A4-induced metastasis. Intracellular S100A4 exists as a homodimer through previously identified, well conserved, predominantly hydrophobic key contacts between the subunits. Here it is shown that mutating just one key residue, phenylalanine 72, to alanine is sufficient to reduce the metastasis-promoting activity of S100A4 to 50% that of the wild type protein, and just 2 or 3 specific mutations reduces the metastasis-promoting activity of S100A4 to less than 20% that of the wild type protein. These mutations inhibit the self-association of S100A4 in vivo and reduce markedly the affinity of S100A4 for at least two of its protein targets, a recombinant fragment of non-muscle myosin heavy chain isoform A, and p53. Inhibition of the self-association of S100 proteins might be a novel means of inhibiting their metastasis-promoting activities.     

Comparing BRIN-BD11 culture producing insulin using different type of microcarriers

This research was conducted to examine the growth profile, growth kinetics, and insulin-secretory responsiveness of BRIN-BD11 cells grown in optimized medium on different types of microcarriers (MCs). Comparisons were made on modified polystyrene (Hillex^® II) and crosslinked polystyrene Plastic Plus (PP) from Solohill Engineering. The cell line producing insulin was cultured in a 25 cm² T-flask as control while MCs based culture was implemented in a stirred tank bioreactor with 1 L working volume. For each culture type, the viable cell number, glucose, lactate, glutamate, and insulin concentrations were measured and compared. Maximum viable cell number was obtained at 1.47 × 10⁵ cell/mL for PP microcarrier (PPMCs) culture, 1.35 × 10⁵ cell/mL Hillex^® II (HIIMCs) culture and 0.95 × 10⁵ cell/mL for T-flask culture, respectively. The highest insulin concentration has been produced in PPMCs culture (5.31 mg/L) compared to HIIMCs culture (2.01 mg/L) and T-flask culture (1.99 mg/L). Therefore overall observation suggested that PPMCs was likely preferred to be used for BRIN-BD11 cell culture as compared with Hillex^® II MCs.     

The Effect of Iron Treatment on Adhesion Molecules in Patients with Iron Deficiency Anemia

The present study was aimed to determine the effect of iron supplementation on levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients with iron deficiency anemia (IDA). In this study, 26 female patients diagnosed with iron deficiency were treated approximately 3 months of oral iron supplementation (99 ± 10 days; ferrous glycine sulfate; 100 mg/day of elemental iron). Levels of sICAM-1 and sVCAM-1 were assessed prior to treatment and after approximately 3 months of treatment and compared with 26 healthy female subjects. A significant increase in sVCAM levels was found in the patients with iron deficiency at the end of the treatment relative to pretreatment levels compared to controls, whereas no significant differences were determined in sICAM levels. In the posttreatment period, no significant change was observed in sICAM levels compared to the pretreatment levels, whereas sVCAM levels decreased. However, after the treatment period, the sVCAM, hemoglobin, mean corpuscular volume (MCV), and serum ferritin levels did not return to the normal range compared to the controls. Pretreatment sVCAM-1 levels were inversely correlated with levels of hemoglobin, hemotocrit, MCV, serum iron, and ferritin. After treatment, the sVCAM-1 levels were negatively correlated with ferritin levels. Levels of sVCAM were significantly higher in patients with IDA than controls. After the treatment period, the sVCAM levels were not completely normalized in patients with IDA compared to controls, regardless of the presence of inadequate levels of hemoglobin, MCV, and serum ferritin. Thus, iron supplementation not only ameliorates anemia, but may also reduce the inflammation markers in cases with IDA.