Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks

Ku protein binds to DNA ends and is a cofactor for the DNA-dependent protein kinase. Both of these components are involved in DNA double-strand break repair, but it has not been clear if they function indirectly, by sensing DNA damage and activating other factors, or if they are more directly involved in the processing and rejoining of DNA breaks. We demonstrate that intermolecular ligation of DNA fragments is highly dependent on Ku under conditions designed to mimic those existing in the cell. This effect of Ku is specific to eukaryotic DNA ligases. Ku protein, therefore, has an activity consistent with a direct role in rejoining DNA breaks and independent of DNA-dependent protein kinase.     

Yeast DNA ligase IV mediates non-homologous DNA end joining

The discovery of homologues from the yeast Saccharomyces cerevisiae of the human Ku DNA-end-binding proteins (HDF1 and KU80) has established that this organism is capable of non-homologous double-strand end joining (NHEJ), a form of DNA double-strand break repair (DSBR) active in mammalian V(D)J recombination. Identification of the DNA ligase that mediates NHEJ in yeast will help elucidate the function of the four mammalian DNA ligases in DSBR, V(D)J recombination and other reactions. Here we show that S. cerevisiae has two typical DNA ligases, the known DNA ligase I homologue CDC9 and the previously unknown DNA ligase IV homologue DNL4. dnl4 mutants are deficient in precise and end-processed NHEJ. DNL4 and HDF1 are epistatic in this regard, with the mutation of each having equivalent effects. dnl4 mutants are complemented by overexpression of Dnl4 but not of Cdc9, and deficiency of Dnl4 alone does not impair either cell growth or the Cdc9-mediated responses to ionizing and ultraviolet radiation. Thus, S. cerevisiae has two distinct and separate ligation pathways.     

Association of a host DNA structure with retroviral integration sites in chromosomal DNA

Integration of retroviral genomes is a site-specific process with respect to the virus but not the host genome. Numerous chromosomal sites and various sequences can be used as targets. Nevertheless, preferential regions and integration patterns have been observed. Using a functional assay, we investigated if host structural DNA elements could be associated with retroviral integration sites. The results were that 9 of 10 distinct retroviral integration events occurred in close proximity of structural elements behaving like intrinsically bent DNA.     

Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs

The budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases. This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs. These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death.     

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.     

Glutamine as a regulator of DNA and protein biosynthesis in human solid tumor cell lines

BACKGROUND: Hip fracture is a preventable cause of disability among elderly people AIM: To study factors associated to hip fractures in Chile. PATIENTS AND METHODS: Patients admitted to seven public hospitals in Chile, with hip fracture not due to major accidents, were considered as cases. To each, at least one age and sex matched hospitalized control, without or neoplastic diseases, was assigned. All patients were subjected to an inquiry, using an instrument devised by the WHO. RESULTS: Two hundred sixty eight cases and 501 controls were studied. Cases and controls has similar educational and labor histories. The right hip was fractures in 47% of cases and the left in the rest. Compared with controls, cases had a higher body mass index, loss of height during life, rate of hysterectomy, incidence of smoking and a lower consumption of diary products. Cases had higher risk of falls inside their homes and controls outside. CONCLUSIONS: The obtained information may contribute to the development of preventive measures and reduce the public health impact of hip fracture.     

Malpractice as a regulator of psychotherapy.

Points out that attempts to expand the malpractice and tort liability of psychologists do not address a major deterrent to claims: the reluctance of clients to sue. Other forms of regulation are needed to compensate for the inability of malpractice to regulate professional psychology. (20 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

The crystal structure of the hyperthermophile chromosomal protein Sso7d bound to DNA

BACKGROUND: Whether herd immunity will occur with widespread Haemophilus influenzae type b (Hib) vaccination in developing countries is dependent on whether the vaccines are capable of reducing carriage in these settings. However, few population-based studies of Hib carriage in developing countries exist. METHODS: To study Hib carriage in the Dominican Republic, we collected nasopharyngeal swab specimens from a population-based sample of 983 children 0 to 47 months old in a periurban area of Santo Domingo. RESULTS: Nasopharyngeal swabs of 76 (7.7%) children were positive for Hib. Hib carriage varied by age group with a low of 1.5% among 0 to 5 month olds, a peak of 12.5% in 6 to 11 month olds and prevalence rates of 6.0, 7.9 and 9.8% among 1-, 2- and 3-year-olds, respectively. Hib carriage was 51% lower among currently breast-fed 6 to 11 month olds than among those not currently breast-fed (18.2% vs. 9.0%; P=0.08). CONCLUSIONS: Infants and young children in Santo Domingo have high rates of Hib carriage, characterized by an early peak in carriage that corresponds with the peak of risk for Hib meningitis. The ability of Hib vaccines to diminish carriage to levels that will effectively reduce transmission and lead to herd immunity in this setting needs to be determined.     

Human autoantibodies reveal titin as a chromosomal protein

Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH2 termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.     

PCR-introduced loop structure as primer in DNA sequencing

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.     

DNA dynamics: different means to a common end?

A ribosomal frameshift is required for the synthesis of an essential component of the yeast telomerase pathway; this and other findings on telomerases from many species raise interesting questions regarding the evolutionary relationship between telomerases and retrotransposons lacking long terminal repeats.     

Protein disulfide isomerase as a regulator of chloroplast translational activation

Light-regulated translation of chloroplast messenger RNAs (mRNAs) requires trans-acting factors that interact with the 5' untranslated region (UTR) of these mRNAs. Chloroplast polyadenylate-binding protein (cPABP) specifically binds to the 5'-UTR of the psbA mRNA and is essential for translation of this mRNA. A protein disulfide isomerase that is localized to the chloroplast and copurifies with cPABP was shown to modulate the binding of cPABP to the 5'-UTR of the psbA mRNA by reversibly changing the redox status of cPABP through redox potential or adenosine 5'-diphosphate-dependent phosphorylation. This mechanism allows for a simple reversible switch regulating gene expression in the chloroplast.     

DNA polymerase-catalyzed addition of nontemplated extra nucleotides to the 3' end of a DNA fragment

Some prokaryotic and eukaryotic DNA polymerases are capable of adding an additional nontemplated nucleotide residue at the 3' end of a DNA fragment (Clark et al., 1987; Clark, 1988). The extra nucleotide at the 3' end of the PCR product has been shown to be a critical factor determining the efficiency of cloning PCR products into plasmids and can affect mutation analyses with a PCR-denaturing gradient gel electrophoresis (DGGE) approach (Pfeiffer and Hu, 1993). In the present work, the ability of various DNA polymerases to add an extra nontemplated nucleotide at the 3' end of DNA was studied. The results show that out of the eight studied enzymes, five can add, with varying efficiencies, an extra nucleotide residue at the 3' end of DNA. Which extra nucleotide is added depends on the terminal residue and the DNA polymerase. Among the enzymes, thermostable Pfu DNA polymerase is found to be the best choice for PCR due to its relatively high fidelity (Scott et al., 1991; Coller, unpublished), and ability to produce blunt-ended DNA fragments. The relationship between the DNA polymerases' ability to add an extra nucleotide and their 3'-->5' exonuclease activity is also discussed.     

Increased nuchal translucency as a marker for fetal chromosomal defects

BACKGROUND: Screening for trisomy 21 (Down's syndrome) by measuring maternal serum alpha-fetoprotein, chorionic gonadotropin, and estriol concentrations and then performing chorionic-villus sampling or amniocentesis identifies approximately 60 percent of fetuses with this disorder. We used ultrasonography to detect increased nuchal translucency and cystic hygroma, which are characteristic features of fetuses with chromosomal defects. METHODS: We performed transvaginal ultrasonography in 10,010 unselected adolescents and women less than 40 years of age with live singleton fetuses at 10 to 15.9 weeks of gestation. Increased fetal nuchal translucency was defined as an area of translucency at least 3 mm in width, and cystic hygromas were defined as septated, fluid-filled sacs in the nuchal region. Subjects whose fetuses had these findings were offered fetal karyotyping. Information on pregnancies, deliveries, and neonates was subsequently obtained from hospital records and national birth and malformation registries. RESULTS: Nuchal translucency or cystic hygroma was seen in 76 fetuses (0.8 percent), of which 18 (24 percent) had an abnormal karyotype. The sensitivity for trisomies 21, 18, and 13 combined was 62 percent (13 of 21 fetuses), and the sensitivity for trisomy 21 alone was 54 percent (7 of 13 fetuses). CONCLUSIONS: The use of transvaginal ultrasonography to detect increased nuchal translucency and cystic hygroma is a sensitive test for fetal aneuploidy. It can be done earlier in pregnancy than serum screening, and it decreases the subsequent need for chorionic-villus sampling or amniocentesis.     

Genetic structure and chromosomal mapping of MyD88

The myeloid differentiation (MyD) marker MyD88 was initially characterized as a primary response gene, upregulated in mouse M1 myeloleukemic cells in response to differentiation induced by interleukin-6. Subsequent analysis revealed that MyD88 possesses a unique modular structure, which consists of an N-terminal "death domain," similar to the intracellular segments of TNF receptor 1 and Fas, and a C-terminal region related to the cytoplasmic domains of the Drosophila morphogen Toll and vertebrate interleukin-1 receptors. In this report we describe the cloning and gene structure of mouse MyD88. The complete coding sequence of mouse MyD88 spans five exons, with the first exon encoding the complete death domain. Zooblot analysis revealed that MyD88 is an evolutionarily conserved gene. MyD88 was localized to the distal region of mouse chromosome 9 by interspecific backcross mapping. The human homolog (hMyD88) was mapped to chromosome 3p22-p21.3 by PCR analysis of a human chromosome 3 somatic cell hybrid mapping panel. Northern blot analysis revealed widespread expression of MyD88 in many adult mouse tissues, and RT-PCR studies detected MyD88 mRNA in T and B cell lines and differentiating embryonic stem cells. The broad expression pattern demonstrates that mouse MyD88 expression is not restricted to cells of myeloid lineage as was originally believed.