The Werner Syndrome Protein Is Distinguished from the Bloom Syndrome Protein by Its Capacity to Tightly Bind Diverse DNA Structures

Loss of Werner syndrome helicase-exonuclease (WRN) or of its homolog Bloom syndrome helicase (BLM) results in different inherited disorders. Whereas Werner syndrome is characterized by premature onset of aging and age-associated diseases, Bloom syndrome involves developmental abnormalities and increased predisposition to diverse malignancies. To identify biochemical differences between WRN and BLM that might contribute to the dissimilar outcomes of their loss, we compared their abilities to unwind and bind in vitro diverse DNA structures. Full-length recombinant WRN and BLM proteins expressed in and purified from Sf9 insect cells unwound to comparable extents and with similar Km values partial DNA duplex, splayed arm DNA and G'2 bimolecular quadruplex DNA. However, WRN resolved bubble DNA ∼25-fold more efficiently than BLM. The two enzymes were mainly distinguished by their contrasting abilities to bind DNA. WRN bound partial duplexes, bubble and splayed arm DNA and G'2 bimolecular and G4 four-molecular quadruplexes with dissociation constants of 0.25 to 25 nM. By contrast, BLM formed substantial complexes with only G4 quadruplex DNA while binding only marginally other DNA structures. We raise the possibility that in addition to its enzymatic activities WRN may act as a scaffold for the assembly on DNA of additional DNA processing proteins

Ecdysteroid Titers in Mated and Unmated \u3ci\u3eDrosophila melanogaster\u3c/i\u3e Females

Radioimmunoassay was used to determine ecdysteroid titers in mated or unmated Drosophila melanogaster females. Whole-body ecdysteroid titers increase after mating and this response is more pronounced after 12–24 hours than it is immediately after mating. In one experiment, females were mated to transgenic males deficient in accessory gland proteins to test whether these peptides mediate the observed increase in female whole-body ecdysteroid titers. Females mated to such transgenic males do not show a pronounced increase in whole-body ecdysteroid titers. The effect of mating on female hemolymph ecdysteroid titers was also investigated. Hemolymph ecdysteroid titers decrease after mating. The ecdysteroid titer change in the hemolymph may result from yolk protein uptake of ecdysteroids into developing vitellogenic oocytes as a consequence of male accessory gland protein stimulation of female oocyte maturation and yolk protein synthesis following mating

Effect of hyperthermia on the survival of normal human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells from normal healthy volunteers were exposed to elevated temperatures of 41-43° for up to 6 hr. Thereafter, the cells were stimulated with phytohemagglutinin in vitro in order to measure indirectly the surviving fraction. DNA replication in heated cells in response to phytohemagglutinin was found to be a sensitive indicator of thermal injury. Exposure to even 40° for 2 hr lowered thymidine incorporation at early time points after phytohemagglutinin stimulation, but the cells were able to recover from thermal injury after exposure for up to 4 hr at 42°. At 43°, exposure for even 1 to 2 hr caused irreversible damage. The changes in thymidine incorporation were not due to changes in endogenous nucleotide pools since parallel changes were observed in DNA polymerase activity. Thus, the heat sensitivity of normal human lymphocytes could be a limiting factor for use of hyperthermia as an adjunct to radiotherapy and chemotherapy of human cancer

DNA replication in X-irradiated human lymphocytes

Human peripheral blood lymphocytes are well-differentiated cells. Ordinarily, they do not divide and are considered to be in the G0 stage of the cell cycle. These cells can be stimulated to undergo DNA replication in culture by mitogens such as phytohemagglutinin. In the present study, we have examined cellular and biochemical events that occur after exposure of lymphocytes to X-irradiation. Irradiation with up to 100 rads, prior to stimulation with phytohemagglutinin, did not interfere with DNA replication. At later periods, DNA replication was inhibited proportionally to the amount of radiation. In comparison to DNA synthesis, the effect of X-irradiation on RNA and protein synthesis in phytohemagglutinin-stimulated lymphocytes was less marked. Furthermore, X-rays did not inhibit either the induction or the continual synthesis of DNA polymerase-α or -β in response to phytohemagglutinin. Kinetic studies with different nucleotide substrates suggest that cellular pools of nucleotides are not significantly altered by χ -irradiation. Thus, the inhibition of DNA synthesis in irradiated cells is likely to be due to damage to the cellular DNA template. The inhibition of DNA synthesis was accompanied by accumulation of cells in the G2 and M stages of the cell cycle, suggesting that inhibition of DNA replication by X -irradiation is a postmitotic event

Frameshift Mutagenesis and Microsatellite Instability Induced by Human Alkyladenine DNA Glycosylase

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine, and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than does the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L-induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing one and two base-pair loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.National Institutes of Health (U.S.) (Grant CA055042)National Institutes of Health (U.S.) (Grant CA115802)National Institutes of Health (U.S.) (Grant ES02109

Altered RECQ Helicase Expression in Sporadic Primary Colorectal Cancers

AbstractDeregulation of DNA repair enzymes occurs in cancers and may create a susceptibility to chemotherapy. Expression levels of DNA repair enzymes have been shown to predict the responsiveness of cancers to certain chemotherapeutic agents. The RECQ helicases repair damaged DNA including damage caused by topoisomerase I inhibitors, such as irinotecan. Altered expression levels of these enzymes in colorectal cancer (CRC) may influence the response of the cancers to irinotecan. Thus, we assessed RECQ helicase (WRN, BLM, RECQL, RECQL4, and RECQL5) expression in primary CRCs, matched normal colon, and CRC cell lines. We found that BLM and RECQL4 mRNA levels are significantly increased in CRC (P = .0011 and P < .0001, respectively), whereas RECQL and RECQL5 are significantly decreased (P = .0103 and P = .0029, respectively). RECQ helicase expression patterns varied between specific molecular subtypes of CRCs. The mRNA and protein expression of the majority of the RECQ helicases was closely correlated, suggesting that altered mRNA expression is the predominant mechanism for deregulated RECQ helicase expression. Immunohistochemistry localized the RECQ helicases to the nucleus. RECQ helicase expression is altered in CRC, suggesting that RECQ helicase expression has potential to identify CRCs that are susceptible to specific chemotherapeutic agents