Mechanism of DNA Methylation: The Double Role of DNA as a Substrate and as a Cofactor

Methylation of cytosine residues in the DNA is one of the most important epigenetic marks central to the control of differential expression of genes. We perform quantum mechanical calculations to investigate the catalytic mechanism of the bacterial HhaI DNA methyltransferase. We find that the enzyme nucleophile, Cys81, can attack C6 of cytosine only after it is deprotonated by the DNA phosphate group, a reaction facilitated by a bridging water molecule. This finding, which indicates that the DNA acts as both the substrate and the cofactor, can explain the total loss of activity observed in an analogous enzyme, thymidylate synthase, when the phosphate group of the substrate was removed. Furthermore, our results displaying the inability of the phosphate group to deprotonate the side chain of serine is in agreement with the total, or the large extent of, inactivity observed for the C81S mutant. In contrast to results from previous calculations, we find that the active site conserved residues, Glu119, Arg163, and Arg165, are crucial for catalysis. In addition, the enzyme-DNA adduct formation and the methyl transfer from the cofactor S-adenosyl-l-methionine are not concerted but proceed via stepwise mechanism. In many of the different steps of this methylation reaction, the transfer of a proton is found to be necessary. To render these processes possible, we find that several water molecules, found in the crystal structure, play an important role, acting as a bridge between the donating and accepting proton groups.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Synthesis and recognition by DNA polymerases of a reactive nucleoside for DNA diversification

The synthesis of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-4-hydrazide having the features of an ambigous base is reported. The recognition of the analogue by DNA polymerases as an incoming triphosphate as well as a template base was investigated. The mutagenic properties was evaluated by PCR. The potential of this new monomer for DNA diversification is illustrated by the reactivity of the nucleobase towards various aldehydes.     

Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymerase

Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.     

DNA Damage Tolerance and a Web of Connections with DNA Repair at Yale

This short article summarizes some of the research carried out recently by my laboratory colleagues on the function of DNA polymerase zeta (polζ) in mammalian cells. Some personal background is also described, relevant to research associations with Yale University and its continuing influence. Polζ is involved in the bypass of many DNA lesions by translesion DNA synthesis and is responsible for the majority of DNA damage-induced point mutagenesis in mammalian cells (including human cells), as well as in yeast. We also found that the absence of this enzyme leads to gross chromosomal instability in mammalian cells and increased spontaneous tumorigenesis in mice. Recently, we discovered a further unexpectedly critical role for polζ: it plays an essential role in allowing continued rapid proliferation of cells and tissues. These observations and others indicate that polζ engages frequently during DNA replication to bypass and tolerate DNA lesions or unusual DNA structures that are barriers for the normal DNA replication machinery.     

Prep-A-Gene: a superior matrix for the purification of DNA and DNA fragments

A new chromatographic matrix, Prep-A-Gene, is described for the isolation and purification of high quality DNA suitable for restriction analysis, ligation, transformation and sequencing protocols. This matrix selectively binds DNA greater than approximately 200 base pairs in length, while RNA, proteins, cellular components, agarose and other contaminants are washed free in minutes. This eliminates the need for time-consuming and laborious RNase treatments, gel extractions and phenol extractions. The DNA that is desorbed from the matrix is available immediately as a substrate for subsequent protocols. DNA purified in this manner exhibits no detectable shearing, even with more fragile chromosomal DNA.     

Synthesis and Recognition by DNA Polymerases of a Reactive Nucleoside for DNA Diversification

Abstract

The synthesis of 1-(2-deoxy-β-D-erythro-pentofuranosyl)imidazole-4-hydrazide having the features of an ambigous base is reported. The recognition of the analogue by DNA polymerases as an incoming triphosphate as well as a template base was investigated. The mutagenic properties was evaluated by PCR. The potential of this new monomer for DNA diversification is illustrated by the reactivity of the nucleobase towards various aldehydes.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

Engineering a DNA damage response without DNA damage

Recent work has achieved the feat of activating the DNA damage checkpoint in the absence of DNA damage, revealing the importance of protein-chromatin associations for the activation, amplification and maintenance of the DNA damage response.     

Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51–200, 271–375, 422–596, and 651–696 of the human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N-terminal 198 amino acid residues of human DNA Topo I. The resulting recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy. Received: 19 July 1999 / Received revision: 10 September 1999 / Accepted: 24 September 1999     

Native and denatured DNA,cross-linked and palindromic DNA and circular covalently-closed DNA analysed by a sensitive fluorometric procedure

Ethidium bromide intercalates duplex DNA with a 25 fold enhancement of its fluorescence. At pH 8 denatured DNA shows about 50% the fluorescence enhancement of duplex DNA due to intramolecular hydrogen-bonding. By raising the pH to 12 short intramolecular duplex regions can be selectively destabilized without altering long duplex DNA. This forms the basis for a sensitive assay for duplex DNA in the presence of denatured DNA. Cross-linked and palindromic DNA differ from normal duplex DNA by their spontaneous renaturation after a heat step with return of fluorescence. Covalently-closed circular DNA is similarly distinguished from open circular DNA.     

Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype

We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in rho0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the rho0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-gamma can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.     

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.     

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.     

Function and disruption of DNA methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

The catalytic domain of Dnmt3a cooperatively multimerizes on DNA forming nucleoprotein filaments. Based on modeling, we identified the interface of Dnmt3a complexes binding next to each other on the DNA and disrupted it by charge reversal of critical residues. This prevented cooperative DNA binding and multimerization of Dnmt3a on the DNA, as shown by the loss of cooperative complex formation in electrophoretic mobility shift assay, the loss of cooperativity in DNA binding in solution, the loss of a characteristic 8- to 10-bp periodicity in DNA methylation and direct imaging of protein-DNA complexes by scanning force microscopy. Non-cooperative Dnmt3a-C variants bound DNA well and retained methylation activity, indicating that cooperative DNA binding and multimerization of Dnmt3a on the DNA are not required for activity. However, one non-cooperative variant showed reduced heterochromatic localization in mammalian cells. We propose two roles of Dnmt3a cooperative DNA binding in the cell: (i) either nucleofilament formation could be required for periodic DNA methylation or (ii) favorable interactions between Dnmt3a complexes may be needed for the tight packing of Dnmt3a at heterochromatic regions. The complex interface optimized for tight packing would then promote the cooperative binding of Dnmt3a to naked DNA in vitro.