GADD45 alpha inhibition of DNMT1 dependent DNA methylation during homology directed DNA repair

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45 alpha. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45 alpha, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45 alpha during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45 alpha increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45 alpha specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45 alpha. Since GADD45 alpha binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair

Lithospheric structure of an Archean craton and adjacent mobile belt revealed from 2-D and 3-D inversion of magnetotelluric data : example from southern Congo craton in northern Namibia

Author Posting. © American Geophysical Union, 2013. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research: Solid Earth 118 (2013): 4378–4397, doi:10.1002/jgrb.50258.Archean cratons, and the stitching Proterozoic orogenic belts on their flanks, form an integral part of the Southern Africa tectonic landscape. Of these, virtually nothing is known of the position and thickness of the southern boundary of the composite Congo craton and the Neoproterozoic Pan-African orogenic belt due to thick sedimentary cover. We present the first lithospheric-scale geophysical study of that cryptic boundary and define its geometry at depth. Our results are derived from two-dimensional (2-D) and three-dimensional (3-D) inversion of magnetotelluric data acquired along four semiparallel profiles crossing the Kalahari craton across the Damara-Ghanzi-Chobe belts (DGC) and extending into the Congo craton. Two-dimensional and three-dimensional electrical resistivity models show significant lateral variation in the crust and upper mantle across strike from the younger DGC orogen to the older adjacent cratons. We find Damara belt lithosphere to be more conductive and significantly thinner than that of the adjacent Congo craton. The Congo craton is characterized by very thick (to depths of  250 km) and resistive (i.e., cold) lithosphere. Resistive upper crustal features are interpreted as caused by igneous intrusions emplaced during Pan-African magmatism. Graphite-bearing calcite marbles and sulfides are widespread in the Damara belt and account for the high crustal conductivity in the Central Zone. The resistivity models provide new constraints on the southern extent of the greater Congo craton and suggest that the current boundary drawn on geological maps needs revision and that the craton should be extended further south.The SAMTEX consortiummembers (Dublin Institute for Advanced Studies, Woods Hole Oceanographic Institution, Council for Geoscience (South Africa), De Beers Group Services, The University of the Witwatersrand, Geological Survey of Namibia, Geological Survey of Botswana, Rio Tinto Mining and Exploration, BHP Billiton, Council for Scientific and Industrial Research (South Africa), and ABB Sweden) are thanked for their funding and logistical support during the four phases of data acquisition. This work is also supported by research grants from the National Science Foundation (EAR-0309584 and EAR-0455242 through the Continental Dynamics Program to R. L. Evans), the Department of Science and Technology, South Africa, and Science Foundation of Ireland (grant 05/RFP/ GEO001to A. G. Jones).2014-02-0

Ubiquitin ligase MKRN1 modulates telomere length homeostasis through a proteolysis of hTERT

Telomere homeostasis is regulated by telomerase and a collection of associatedproteins. Telomerase is, in turn, regulated by post-translational modifications of the rate-limiting catalytic subunit hTERT. Here we show that disruption of Hsp90 by geldanamycin promotes efficient ubiquitination and proteasome-mediated degradation of hTERT. Furthermore, we have used the yeast two-hybrid method to identify a novel RING finger gene (MKRN1) encoding an E3 ligase that mediates ubiquitination of hTERT. Overexpression of MKRN1 in telomerase-positive cells promotes the degradation of hTERT and decreases telomerase activity and subsequently telomere length. Our data suggest that MKRN1 plays an important role in modulating telomere length homeostasis through a dynamic balance involving hTERT protein stability

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes ( and DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, , , and . We found that for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5\u27-triphosphate binding to DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors

DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells

A Survey of FUSE and HST Sightlines through High-Velocity Cloud Complex C

Using archival Far Ultraviolet Spectroscopic Explorer (FUSE) and Hubble Space Telescope (HST) data, we have assembled a survey of eight sightlines through high-velocity cloud Complex C. Abundances of the observed ion species vary significantly for these sightlines, indicating that Complex C is not well characterized by a single metallicity. Reliable metallicities based on [OI/HI] range from 0.1-0.25 solar. Metallicities based on [SII/HI] range from 0.1-0.6 solar, but the trend of decreasing abundance with HI column density indicates that photoionization corrections may affect the conversion to [S/H]. We present models of the dependence of the ionization correction on HI column density; these ionization corrections are significant when converting ion abundances to elemental abundances for S, Si, and Fe. The measured abundances in this survey indicate that parts of the cloud have a higher metallicity than previously thought and that Complex C may represent a mixture of ``Galactic fountain'' gas with infalling low-metallicity gas. We find that [S/O] and [Si/O] have a solar ratio, suggesting little dust depletion. Further, the measured abundances suggest an over-abundance of O, S, and Si relative to N and Fe. The enhancement of these alpha-elements suggests that the bulk of the metals in Complex C were produced by Type II supernovae and then removed from the star-forming region, possibly via supernovae-driven winds or tidal stripping, before the ISM could be enriched by N and Fe.Comment: 50 pages, 17 figures. Accepted to ApJ. Previous version was the first submitted manuscrip

High-coverage methylation data of a gene model before and after DNA damage and homologous repair

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles

DNA-protein kinase catalytic subunit-interacting protein KIP binds telomerase by interacting with human telomerase reverse transcriptase

Telomere homeostasis, a process that is essential for continued cell proliferation and genomic stability, is regulated by endogenous telomerase and a collection of associated proteins. In this paper, a protein called KIP ( previously reported as a protein that binds specifically to DNA-dependent protein kinase), has been identified as a telomerase-regulating activity based on the following pieces of evidence. First, complexes between KIP and the catalytic subunit of telomerase ( hTERT) were identified using the yeast two-hybrid technique. Second, antibodies specific to KIP immunoprecipitate human telomerase in cell-free extracts. Third, immunolocalization experiments demonstrate that KIP is a nuclear protein that co-localizes with hTERT in cells. Fourth, KIP binds to hTERT both in vitro and in vivo in the absence of human telomerase RNA or telomeric DNA, thus defining the catalytic subunit of telomerase as the site of physical interaction. Fifth, co-immunoprecipitation experiments suggest that KIP-hTERT complexes form readily in cells and that overexpression of KIP in telomerase-positive cells increases endogenous telomerase activity. Finally, continued overexpression of KIP ( 60 population doublings) resulted in cells with elongated telomeres; thus, KIP directly or indirectly stimulates telomerase activity through hTERT and contributes to telomere lengthening. The collective data in this paper suggest that KIP plays a positive role in telomere length maintenance and/or regulation and may represent a novel target for anti-cancer drug development