Induced crossing-over in Drosophila melanogaster germ cells of DNA repair-proficient and repair-deficient (mei-9L1) males following larval feeding with 5-azacytidine and mitomycin C

Pontecorvo, Giovanni; Avitabile, Angela; Esposito, Gabriella; Migliaccio, Giovanni; Carfagna, M.

doi:10.1016/0027-5107(92)90176-3

Cited by 7 publications

(5 citation statements)

References 30 publications

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“…A precedent for cellular mechanisms that reverse covalent protein-DNA adducts has been reported in eukaryotes (14,26,40); therefore, it seems likely that enzymatic (or other) means exist to reverse (or repair) the DNMT1-DNA adducts. The finding that aza-dC stimulates recombination in the fruit fly (24,25) further suggests that recombination repair may be the adduct reversal that we report here. These reversal pathways are of obvious importance to the application of hypomethylating drugs in the treatment of cancer.…”

Section: Discussionsupporting

confidence: 73%

Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

Liu

Wang

Cantemir

et al. 2003

Molecular and Cellular Biology

136

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While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.In eukaryotes, DNA methylation is an epigenetic encryption system that is essential for proper gene regulation (for reviews see references 2, 4, 7, 23, 29, and 30). Defects in methylation lead to diverse disorders from mental retardation to immune deficiencies, and there is particularly strong evidence that methylation defects create a favorable environment for malignant transformation (2, 3). Pharmacologic alterations in methylation of specific genes have also been correlated with tumor response to chemotherapy and patient survival; thus, methylation regulation and the enzymes that catalyze the process represent important areas for treating cancer (2, 30).The enzymatic machinery that mediates methylation involves a number of DNA (cytosine-5) methyltransferase (DNMT) isoforms, including DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b (and a host of DNMT3 splice variants) (4, 29, 30). Dnmt1, Dnmt3a, and Dnmt3b are independent genes and essential; embryos lacking both copies of Dnmt1 or Dnmt3b die before birth, whereas Dnmt3a-nulls survive about 4 weeks (18, 21). Heterozygous mutants appear normal and are fertile (18, 21). The murine DNMT3a and -3b enzymes appear to possess de novo methylation activity (based upon plasmid methylation), and there is evidence that they act on different DNA targets in vivo (12). No transmethylase activity has been found with DNMT2, and biallelic deletions appear to possess normal methylation patterns (8,22). How different DNMTs ar...

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Section: Discussionsupporting

confidence: 73%

Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

Liu

Wang

Cantemir

et al. 2003

Molecular and Cellular Biology

136

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“…The detection of methylcytosine in the DNA of D.melanogaster is in agreement with several observations: (i) D.melanogaster contains a gene for a putative DNA methyltransferase (Hung et al ., 1999; Tweedie et al ., 1999); (ii) proteins that resemble methylcytosine binding proteins have been identified in D.melanogaster (Hung et al ., 1999; Tweedie et al ., 1999); (iii) other insects also contain methylcytosine (Adams et al ., 1979; Deobagkar et al . , 1982; Patel and Gopinathan, 1987; Field, 1989; Devajyothi and Brahmachari, 1992; Tweedie et al ., 1999); (iv) the artificial introduction of high levels of DNA methylation by transgenic expression of the murine Dnmt3a DNA methyltransferase in D.melanogaster leads to lethality during development (Lyko et al ., 1999), indicating that methylation causes a biological response; and (v) 5‐azacytidine is cytotoxic in D.melanogaster and leads to increased levels of recombination (Katz, 1985; Osgood and Seward, 1989; Pontecorvo et al ., 1992). This drug is toxic because it forms covalent complexes between the DNA and DNA methyltransferases (Ferguson et al ., 1997; Jackson‐Grusby et al ., 1997) that require recombination for repair (Barbe et al ., 1986; Bhagwat and Roberts, 1987; Lal et al ., 1988).…”

Section: Resultsmentioning

confidence: 99%

DNA of Drosophila melanogaster contains 5-methylcytosine

2000

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It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5-methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly speci®c and sensitive assay to detect the presence of 5-methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5-methylcytosine puri®ed by HPLC and, for detection by 1D-and 2D-TLC, radiolabeled using deoxynucleoside kinase and [g-32 P]ATP. Using this assay, we show here that 5-methylcytosine occurs in the DNA of D.melanogaster at a level of~1 in 1000±2000 cytosine residues in adult¯ies. DNA methylation is detectable in all stages of D.melanogaster development.

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“…The detection of methylcytosine in the DNA of D.melanogaster is in agreement with several observations: (i) D.melanogaster contains a gene for a putative DNA methyltransferase (Hung et al, 1999;Tweedie et al, 1999); (ii) proteins that resemble methylcytosine binding proteins have been identi®ed in D.melanogaster (Hung et al, 1999;Tweedie et al, 1999); (iii) other insects also contain methylcytosine (Adams et al, 1979;Deobagkar et al, 1982;Patel and Gopinathan, 1987;Field, 1989;Devajyothi and Brahmachari, 1992;Tweedie et al, 1999); (iv) the arti®cial introduction of high levels of DNA methylation by transgenic expression of the murine Dnmt3a DNA methyltransferase in D.melanogaster leads to lethality during development (Lyko et al, 1999), indicating that methylation causes a biological response; and (v) 5-azacytidine is cytotoxic in D.melanogaster and leads to increased levels of recombination (Katz, 1985;Osgood and Seward, 1989;Pontecorvo et al, 1992). This drug is toxic because it forms covalent complexes between the DNA and DNA methyltransferases (Ferguson et al, 1997;Jackson-Grusby et al, 1997) that require recombination for repair (Barbe et al, 1986;Bhagwat and Roberts, 1987;Lal et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

DNA of Drosophila melanogaster contains 5-methylcytosine

Gowher

Leismann

Jeltsch

2000

Biochemical Society Transactions

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820 Sequence-specific nuclear matrix proteins share rod-domain with intermediate filaments. A.R. Goloudina, A.P. Voronin, A.S. Kukalev, N.I. Enukashvily 194064, Russia, St.Petersburg, Tichoretskii av.4 Lamins, which are the major and well-investigated components of the nuclear matrix (NM), belong to the 5-th class of the wide family of the intermediate filament composing proteins. Lamins are a component of the inner NM as well as the NM outer partlamina. Nature of the main fibrillar component of the NM remains obscure constant attempts notwithstanding. From its morphological appearance the main NM proteins belong to the group of intermediate filaments; still all the antibodies (AB) against different proteins from this group stain only part of the filaments or filaments fragments. Lamins DNA binding properties are unquestionable but the sequence-specificity is not distinct. Mouse Membrane-associated Telomere Binding Protein (mMTBP) was found in NM preparations with the AB against MTBP and high affinity binding to (T2AG3)135 vertebrate telomere fragment TRF2/mMTBP localised near NE and bind telomers which are to be in contact with NE. TRF2/mMTBP possesses rod-domain of intermediate filaments proteins according to the reactivity with the correspondent AB and computer analysis of the TFRZ sequence published. Interaction between NM and pericentromeric alpha satellite DNA (alfa-DNA) caused by NM proteins specific in binding. The results of 2-D electrophoresis of DNA-protein complexes formed by aDNA from the chromosome 21 centromeric region ( without CENP-B box) and NM proteins, hyper-shift assay and immunoblotting showed that these complexes include proteins with Mr approx. 80 and 70 kD which are neither CENP-B nor lamins A/C and B. The major protein of the complex of 70 kD is not cloned yet but also contains rod-domain according to the reactivity with the correspondent AB So at least several NM proteins with more or less determined sequencebinding specificity share rod-domain but have different DNA-binding domains.

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Induced crossing-over in Drosophila melanogaster germ cells of DNA repair-proficient and repair-deficient (mei-9L1) males following larval feeding with 5-azacytidine and mitomycin C

Cited by 7 publications

References 30 publications

Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

Endogenous Assays of DNA Methyltransferases: Evidence for Differential Activities of DNMT1, DNMT2, and DNMT3 in Mammalian Cells In Vivo

DNA of Drosophila melanogaster contains 5-methylcytosine

DNA of Drosophila melanogaster contains 5-methylcytosine

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