Questions and Answers (1) View all
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Answer added in Western Blot15 I am trying to use western blots to detect methylated histones, but I'm getting really inconsistent results. Is anyone else trying to do this?My student encountered the same problem, which in the end was solved by doing an acid extraction of histones, described in his article, which appears ... [more]
Publications (52) View all
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Article: RNAi screening uncovers Dhx9 as a modifier of ABT-737 resistance in an Eμ-myc/Bcl-2 mouse model.
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ABSTRACT: ABT-737 is a promising chemotherapeutic agent that promotes apoptosis by acting as a selective BH3-mimetic to neutralize Bcl-2-like family members. One shortcoming with its use is that Mcl-1, a member of the Bcl-2 family, is poorly inhibited by ABT-737 and, thus a major cause of resistance. We performed a shRNA-based drop-out screen to identify novel genes and pathways that could reverse resistance to ABT-737 treatment in Eμ-myc/Bcl-2 lymphoma cells engineered to rely on endogenous Mcl-1 for survival. Several drug-sensitive shRNAs were identified that were selectively depleted in the presence of ABT-737. Of these, two independent shRNAs targeting the RNA/DNA helicase Dhx9 were found to sensitize lymphomas to ABT-737 to an extent comparable to control Mcl-1 shRNAs. Although Dhx9 suppression sensitized both mouse and human cells to ABT-737 treatment, it did so without altering MCL-1 levels. Rather, loss of Dhx9 appeared to activate a p53-dependent apoptotic program, through aggravation of replicative stress, which was found to be both necessary and sufficient for the ABT-737-shDhx9 synthetic lethal relationship.Blood 02/2013; · 9.90 Impact Factor -
Chapter: Synergy Between DNA Replication and Repair Mechanisms
09/2011; , ISBN: 978-953-307-649-2 -
Article: Transient dsDNA breaks during pre-replication complex assembly.
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ABSTRACT: Initiation of DNA replication involves the ordered assembly of the multi-protein pre-replicative complex (pre-RC) during G(1) phase. Previously, DNA topoisomerase II (topo II) was shown to associate with the DNA replication origin located in the lamin B2 gene locus in a cell-cycle-modulated manner. Here we report that activation of both the early-firing lamin B2 and the late-firing hOrs8 human replication origins involves DNA topo II-dependent, transient, site-specific dsDNA-break formation. Topo IIbeta in complex with the DNA repair protein Ku associates in vivo and in vitro with the pre-RC region, introducing dsDNA breaks in a biphasic manner, during early and mid-G(1) phase. Inhibition of topo II activity interferes with the pre-RC assembly resulting in prolonged G(1) phase. The data mechanistically link DNA topo IIbeta-dependent dsDNA breaks and the components of the DNA repair machinery with the initiation of DNA replication and suggest an important role for DNA topology in origin activation.Nucleic Acids Research 08/2009; 37(17):5714-24. · 8.03 Impact Factor -
Article: Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts.
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ABSTRACT: DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.Journal of Cellular Biochemistry 07/2008; 104(3):773-84. · 2.87 Impact Factor -
Article: Ku is involved in cell growth, DNA replication and G1-S transition.
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ABSTRACT: The Ku protein (Ku70-Ku80) is involved in various genome-maintenance processes such as DNA replication and repair, telomere maintenance, and chromosomal stability. We previously found that Ku80 is implicated in the loading of members of the pre-replicative complex (pre-RC) onto replication origins. Here, we report that acute reduction of Ku80 to 10% of its normal levels leads to impaired DNA replication and activation of a replication stress checkpoint. In the absence of Ku80, decreased levels of the initiator proteins Orc1 and Orc6 as well as reduced chromatin binding of Orc1, Orc4 and Cdc45 were observed, leading to decreased origin firing, whereas Orc2 and Orc3 were unaffected. Prolonged perturbation of DNA replication caused the block of cell-cycle progression in late G1 phase with low Cdk2 activity due to increased p21 expression and decreased Cdc25A and Cdk2 levels. The data suggest the interplay between the DNA-replication and cell-cycle machineries and shed light on a new role of Ku in G1-S transition.Journal of Cell Science 04/2008; 121(Pt 5):590-600. · 6.11 Impact Factor
