Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

Telomere homeostasis is regulated by telomerase and a collection of associated proteins. Telomerase is, in turn, regulated by post-translational modifications of the ratelimiting 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.

The cytosine analog 5-aza-2-deoxycytidine is a potent inhibitor of DNA methyltransferase. Its cytotoxicity has been attributed to several possible mechanisms including reexpression of growth suppressor genes and formation of covalent adducts between DNA methyltransferase and 5-aza-2-deoxycytidine-substituted DNA which may lead to steric inhibition of DNA function. In this study, we use a panel of human breast cancer cell lines as a model system to examine the relative contribution of two mechanisms, gene reactivation and adduct formation. Estrogen receptor-negative cells, which have a hypermethylated estrogen receptor gene promoter, are more sensitive than estrogen receptor-positive cells and underwent apoptosis in response to 5-aza-2-deoxycytidine. For the first time, we show that reactivation of a gene silenced by methylation, estrogen receptor, plays a major role in this toxicity in one estrogen receptor-negative cell line as treatment of the cells with anti-estrogen-blocked cell death. However, drug sensitivity of other tumor cell lines correlated best with increased levels of DNA methyltransferase activity and formation DNA⅐DNA methyltransferase adducts as analyzed in situ. Therefore, both reexpression of genes like estrogen receptor and formation of covalent enzyme⅐ DNA adducts can play a role in 5-aza-2-deoxycytidine toxicity in cancer cells.Current studies suggest that DNA methyltransferase (DNA MTase), 1 the enzyme that methylates cytosines that are 5Ј to guanosines, plays a role in human carcinogenesis. In general, the level of DNA MTase activity is elevated significantly in neoplastic cells compared with normal cells (1). Moreover, increased enzyme activity is characteristic of the progression of both colon and lung cancer (2, 3). Studies demonstrate that overexpression of DNA MTase leads to the tumorigenic conversion of NIH3T3 cells (4), whereas decreasing the levels of DNA MTase through a combination of genetic and pharmacologic means drastically reduces the incidence of colonic adenomas in the Apc min mouse model of colon carcinogenesis (5). These studies provide substantial evidence for the involvement of DNA MTase in oncogenesis.There are at least two potential mechanisms by which DNA MTase may influence oncogenicity. Elevated levels of DNA methylation may lead to increased frequency of C to T transition mutations derived from deamination of methylcytosine (6). Alternatively, increased DNA MTase may play a role in the establishment of altered patterns of methylation at CpG island sequences found in the 5Ј region of genes involved in growth control and tumor progression (7). For example, aberrant hypermethylation of CpG islands in cancer cells has been implicated in the transcriptional inactivation of the Rb, p16, estrogen receptor (ER), E-cadherin, and glutathione S-transferase Pi genes (8 -12).These studies have sparked a renewed interest in the use of DNA MTase inhibitors such as the cytosine analogs 5-azacytidine and 5-aza-2Ј-deoxycytidine (5-aza-dC) in the treatment of human cancers. In vitro...

The distribution of eukaryotic DNA topoisomerase I in the cell has been analyzed at four levels: (i) at the level of the nuclear matrix; (ii) at the cytological level by immunofluorescence of whole cells; (iii) at the electron microscopic level using the protein A/colloidal gold technique; and (iv) at the level of DNA to identify in situ the sequence upon which topoisomerase I is catalytically active. Although topoisomerase I is clearly distributed non‐randomly in the nucleus, the unique distribution of the enzyme is not related to the nuclear matrix. The data support the conclusion that topoisomerase I is heavily concentrated in the nucleolus of the cell; furthermore, particular regions within the nucleolus are depleted of topoisomerase. A technique has been developed which allows isolation and analysis of the cellular DNA sequences covalently attached to topoisomerase. Ribosomal DNA sequences are at least 20‐fold enriched in topoisomerase/DNA complexes isolated directly from a chromosomal setting, relative to total DNA. This is the first direct evidence that topoisomerase I is catalytically active on ribosomal DNA in vivo.

A gel electrophoresis DNA-binding assay was used to detect proteins from herpes simplex virus type 1infected and uninfected cells that specifically bind the upstream region of immediate-early (IE) gene 3. The assay is based on the altered electrophoretic mobility of DNA-protein complexes relative to that of free DNA in native gels. A series of end-labeled overlapping DNA fragments spanning a region from-272 to +27 (relative to the 5' terminus of the IE gene 3 mRNA) were used as probes. Two complexes were identified (referred to as A and B) which were driven by different protein factors. Formation of the A complex required (i) infected-cell proteins extracted at any time from 2 to 16 h postinfection; (ii) a 0.5 to 1 M NaCl extract of infected cells, and (iii) a DNA probe that contained the sequences from-4 to +27 (relative to the 5' terminus of IE gene 3 mRNA). The protein that drove the formation of the A complex is not related to transcription factors TFIIIA or Spl or their cognate binding domains since neither the 5S RNA gene nor the GC box of simian virus 40 could compete for proteins that induced formation of the A complex. Through the use of monoclonal antibodies, the complex was shown to contain the IE gene 3 product, ICP4. A more detailed localization of the DNA-binding site in vitro by using chemical footprinting revealed that binding occurs over the sequence from-10 to +3 relative to the mRNA terminus. The binding of ICP4 to its own transcription start

The activity of the endogenous DNA topoisomerase type I (EC 5.99.1.2) can be quantified in situ by determining how efficiently the enzyme is trapped in a covalent complex with DNA upon lysis of nuclei with detergents. In this way, we can measure relative levels of topoisomerase binding to DNA at native sites in chromatin. Since the majority of topoisomerase I is localized in the nucleolus at rRNA genes, we have evaluated how low levels of actinomycin D, which terminate transcription of rRNA genes, affect the activity of topoisomerase I. In vivo, as well as in vitro with purified topoisomerase I, we have found that drug treatment extends the half-life of the covalent topoisomerase-DNA complex. Actinomycin D stabilizes the nicked intermediate in the cleavage and resealing reaction but otherwise does not significantly alter the strand-passing ability of topoisomerase I. Sequence-specific cleavages by topoisomerase I were stimulated by actinomycin D at identical sequences recognized by the enzyme in the absence of drug. The localization of topoisomerase I in the nucleolus, coupled with the observation that transcription in this organelle is highly sensitive to actinomycin D and camptothecin treatment, leads us to propose that topoisomerase I contributes to actinomycin D inhibition of transcription.A number of reports have suggested that type I DNA topoisomerase (topoisomerase I; EC 5.99.1.2) is involved in transcription based on its association with actively transcribed genes in chromatin (1-6). This association is clearly evident for rRNA genes in animals, yeast, and Tetrahymena (2,3,7). The enzyme is acting catalytically at genes characterized by a high rate of transcription and the heaviest enrichment of topoisomerase I is seen cytologically within the nucleolus (2). Topoisomerase I makes a transient singlestrand break in the sugar-phosphate backbone of DNA (for reviews, see refs. 8 and 9), which introduces a site of rotational freedom in the template; thus, topoisomerase action may provide a swivel point to facilitate entry and/or progression of the bulky transcriptional apparatus. Alternatively, when nascent RNA chains are hybridized to the one strand of template, the topology changes and topoisomerase I may be required to return to (or adjust) the topological ground state. Studies of yeast topoisomerase I mutants suggest that topoisomerase I is not an essential gene (10, 11); however, it seems that topoisomerase II (EC 5.99.1.3) is complementing the defect since topoisomerase II (like topoisomerase I) has been associated with transcriptionally active regions in chromatin (ref. 12; M.T.M. and V. Mehta, unpublished data) and topoisomerase I and II double mutants display a defect in rRNA transcription (7).A better understanding of the involvement of topoisomerase I in transcription can be obtained by analyzing the activity of topoisomerase I catalyzed reactions in a chromosomal setting. The covalent intermediate is a functional reaction intermediate in the process of breaking and rejoining the DNA substra...

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...