1,721,070 research outputs found
Transcription influences repair-induced DNA methylation
No full textThis work is aimed at the dissection of the molecular mechanism(s) linking DNA damage and gene silencing. To this end, we have developed a genetic system that allows a rapid assessment of homologous-directed repair (HR) of an unique DNA double strand break (DSB). Briefly, we induced a DBS in the genome of HeLa or mouse embryonic stem (ES) cells using the I-SceI restriction endonuclease. Homologous recombination repair by gene conversion, initiated at the site of the double strand break, converts 2 inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP) in an intact functional gene. The efficiency of HR, under our conditions, is approximately 2%–4% and can be easily quantified by analyzing GFP+ cells. Half of these recombinants expressed GFP poorly, because GFP gene was silenced. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since HeLa DR-GFP treatment with 5-aza-2’-deoxycytidine, a DNA demethylating drug, significantly increased the fraction of GFP expressing cells. Methylation did not alter recombination frequency in both cell types. ES cells deficient in DNA methyl-transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Bisulfite analysis of GFP DNA molecules revealed that approximately half of the HR repaired molecules were de novo methylated, principally at the 3’-end of the DSB in a range of ~300bp. The other half GFP molecules were hypomethylated. Uncleaved and non-homologous repaired molecules did not show changes of the methylation profile. DNA methyl-transferase 1 bound specifically to HR GFP DNA, as revealed by chromatin immunoprecipitation and RNA analysis. HR induced novel methylation profiles on top of the old patterns and contributed to the silencing of GFP expression. Inhibition of transcription by -amanitin for a very short period (6-24 h during ISceI cleavage) significantly reduced the frequency of recombination. Surprisingly, the 2 classes of recombinants were better separated in terms of GFP expression. Methylation analysis showed that the methylated molecules were hypermethylated, whereas the hypomethylated GFP gene molecules were un-methylated, relative to the untreated samples. Taken together, our data support a mechanistic link between HR, DNA methylation and transcription. We propose that stalled RNA polymerase molecules slow down homologous recombination by interfering possibly with DNA polymerase complex or strand invasion. At the same time, RNA polymerase II transcription complex signals to DNMT1 the coding strand and facilitates strand selective DNA methylation. Overall, these data highlight a new and unexpected opportunity to understand the mechanisms of silencing of damaged and repaired genes
Escherichia Coli mutations that block transcription termination by phage HK022 Nun protein
No full textThe nun gene product of the lambdoid coliphage HK022 provokes premature transcription termination at, or near, the phage λ nut sites. Termination by Nun and antitermination by λ N protein both require the nut sites and Escherichia coli NusA, NusB and NusE proteins. To characterize further the host requirements for Nun termination, we selected host mutations that blocked termination at λ nutR. In addition to mutations in nusA, nusB and nusE, we obtained mutations in rpoC, encoding the RNA polymerase β′ subunit. The nusA and rpoC mutations suppressed Nun termination but not antitermination by λ N function. The mutations antagonized Nun only at λ nutR; termination at λ nutL occurred in all the mutant strains. Thus, nutL is not functionally equivalent to nutR. We conclude that the host requirements for Nun termination overlap but are not identical with those for N antitermination, and, in particular, that the β′ subunit of RNP may be Nun-specific
Lambda nutR mutations convert HK022 Nun protein from a transcription termination factor to a suppressor of termination
No full textThe Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type
The Biological Functions of A-Kinase Anchor Proteins.
No full textcAMP-dependent protein kinase is targeted to discrete subcellular locations by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs). Localization recruits protein kinase A (PKA) holoenzyme close to its substrate/effector proteins, directing and amplifying the biological effects of cAMP signaling.
AKAPs include two conserved structural modules: (i) a targeting domain that serves as a scaffold and membrane anchor; and (ii) a tethering domain that interacts with PKA regulatory subunits. Alternative splicing can shuffle targeting and tethering domains to generate a variety of AKAPs with different targeting specificity. Although AKAPs have been identified on the basis of their interaction with PKA, they also bind other signaling molecules, mainly phosphatases and kinases, that regulate AKAP targeting and activate other signal transduction pathways.
We suggest that AKAP forms a "transduceosome" by acting as an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The transduceosome amplifies cAMP and other signals locally and, by stabilizing and reducing the basal activity of PKA, it also exerts long-distance effects. The AKAP transduceosome thus optimizes the amplitude and the signal/noise ratio of cAMP-PKA stimuli travelling from the membrane to the nucleus and other subcellular compartments
The Biological Functions of A-Kinase Anchor Proteins.
No full textcAMP-dependent protein kinase is targeted to discrete subcellular locations by a family of specific anchor proteins (A-kinase anchor proteins, AKAPs). Localization recruits protein kinase A (PKA) holoenzyme close to its substrate/effector proteins, directing and amplifying the biological effects of cAMP signaling.
AKAPs include two conserved structural modules: (i) a targeting domain that serves as a scaffold and membrane anchor; and (ii) a tethering domain that interacts with PKA regulatory subunits. Alternative splicing can shuffle targeting and tethering domains to generate a variety of AKAPs with different targeting specificity. Although AKAPs have been identified on the basis of their interaction with PKA, they also bind other signaling molecules, mainly phosphatases and kinases, that regulate AKAP targeting and activate other signal transduction pathways.
We suggest that AKAP forms a "transduceosome" by acting as an autonomous multivalent scaffold that assembles and integrates signals derived from multiple pathways. The transduceosome amplifies cAMP and other signals locally and, by stabilizing and reducing the basal activity of PKA, it also exerts long-distance effects. The AKAP transduceosome thus optimizes the amplitude and the signal/noise ratio of cAMP-PKA stimuli travelling from the membrane to the nucleus and other subcellular compartments
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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