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PubMed1994

Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases.

Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes.

Amino Acid Sequence↗

PubMed1995

Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.

We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.

Arabidopsis↗

PubMed1996

Cloning and analysis of the genes encoding the type IIS restriction-modification system HphI from Haemophilus parahaemolyticus.

The genomic region encoding the type IIS restriction-modification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5'-GGTGA-3'/5'-TCACC-3') from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.Hindill at overlapping sites, suggesting that the adenine methyltransferase modifies the 3'-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete gaIE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

Amino Acid Sequence↗

PubMed1997

Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase.

The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.

Amino Acid Sequence↗

PubMed1998

Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci.

It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.

Animals↗

PubMed1998

Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences.

We describe a new primer design strategy for PCR amplification of unknown targets that are related to multiply-aligned protein sequences. Each primer consists of a short 3' degenerate core region and a longer 5' consensus clamp region. Only 3-4 highly conserved amino acid residues are necessary for design of the core, which is stabilized by the clamp during annealing to template molecules. During later rounds of amplification, the non-degenerate clamp permits stable annealing to product molecules. We demonstrate the practical utility of this hybrid primer method by detection of diverse reverse transcriptase-like genes in a human genome, and by detection of C5DNA methyltransferase homologs in various plant DNAs. In each case, amplified products were sufficiently pure to be cloned without gel fractionation. This COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy has been implemented as a computer program that is accessible over the World Wide Web (http://blocks.fhcrc.org/codehop.html) and is directly linked from the BlockMaker multiple sequence alignment site for hybrid primer prediction beginning with a set of related protein sequences.

Amino Acid Sequence↗

PubMed1999

Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae.

Several mutants ( spb1 - spb7 ) have been previously identified as cold-sensitive extragenic suppressors of loss-of-function mutations in the poly(A)(+)-binding protein 1 of Saccharomyces cerevisiae. Cloning, sequence and disruption analyses revealed that SPB1 (YCL054W) encodes an essential putative S -adenosylmethionine-dependent methyltransferase. Polysome analyses showed an under-accumulation of 60S ribosomal subunits in the spb1-1 mutant and in a strain genetically depleted of Spb1p. Northern and primer extension analyses indicated that this was due to inhibition of processing of the 27SB precursors, which results in depletion of the mature 25S and 5.8S rRNAs. At later time points of Spb1p depletion, the stability of 40S ribosomal subunits is also affected. These results suggest that Spb1p is involved in 60S ribosomal subunit biogenesis and associates early with the pre-ribosomes. Consistent with this, hemagglutinin epitope-tagged Spb1p localizes to the nucleus with nucleolar enrichment. Despite the expected methyltransferase activity of Spb1p, global methylation of pre-rRNA is not affected upon Spb1p depletion. We propose that Spb1p is required for proper assembly of pre-ribosomal particles during the biogenesis of 60S ribosomal subunits.

Amino Acid Sequence↗

PubMed2000

Expression of ZmMET1, a gene encoding a DNA methyltransferase from maize, is associated not only with DNA replication in actively proliferating cells, but also with altered DNA methylation status in cold-stressed quiescent cells.

A cDNA fragment encoding part of a DNA methyltransferase was isolated from maize. The putative amino acid sequence identically matched that deduced from a genomic sequence in the database (accession no. AF063403), and the corresponding gene was designated as ZmMET1. Bacterially expressed ZmMET1 actively methylated DNA in vitro. Transcripts of ZmMET1 could be shown to exclusively accumulate in actively proliferating cells of the meristems of mesocotyls and root apices, suggesting ZmMET1 expression to be associated with DNA replication. This was confirmed by simultaneous decrease of transcripts of ZmMET1 and histone H3, a marker for DNA replication, in seedlings exposed to wounding, desiccation and salinity, all of which suppress cell division. Cold stress also depressed both transcripts in root tissues. In contrast, however, accumulation of ZmMET1 transcripts in shoot mesocotyls was not affected by cold stress, whereas those for H3 sharply decreased. Such a differential accumulation of ZmMET1 transcripts was consistent with ZmMET1 protein levels as revealed by western blotting. Expression of ZmMET1 is thus coexistent, but not completely dependent on DNA replication. Southern hybridization analysis with a methylation-sensitive restriction enzyme revealed that cold treatment induced demethylation of DNA in the Ac/Ds transposon region, but not in other genes, and that such demethylation primarily occurred in roots. These results suggested that the methylation level was decreased selectively by cold treatment, and that ZmMET1 may, at least partly, prevent such demethylation.

Blotting, Southern↗

PubMed2000

DNA methylation at the CfrBI site is involved in expression control in the CfrBI restriction-modification system.

We have previously found that genes of the CFR:BI restriction-modification (R-M) system from Citrobacter freundii are oriented divergently and that their promoter regions overlap. The overlapping promoters suggest regulation of gene expression at the transcriptional level. In this study the transcription regulation of CFR:BI R-M genes was analyzed in vivo and in vitro in Escherichia coli. It was shown that in the presence of CFR:BI methyltransferase (M.CFR:BI), cell galactokinase activity decreases 10-fold when the galactokinase gene (galK) is under the control of the cfrBIM promoter and increases 20-fold when galK is under the control of the cfrBIR promoter. The CFR:BI site, proven to be unique for the entire CFR:BI R-M gene sequence, is located in the -35 cfrBIM promoter region and is in close vicinity of the -10 cfrBIR promoter region. A comparison of the cfrBIM and the cfrBIR promoter activities in the in vitro transcription system using methylated and unmethylated DNA fragments as templates demonstrated that the efficiency of CFR:BI R-M gene transcription is regulated by enzymatic modification at the N-4-position of cytosine bases of the CFR:BI site by M.CFR:BI. From the results of the in vivo and in vitro experiments we suggest a new model of gene expression regulation in type II R-M systems.

Base Sequence↗

PubMed2002

Genomic repeats, genome plasticity and the dynamics of Mycoplasma evolution.

Mycoplasmas evolved by a drastic reduction in genome size, but their genomes contain numerous repeated sequences with important roles in their evolution. We have established a bioinformatic strategy to detect the major recombination hot-spots in the genomes of Mycoplasma pneumoniae, Mycoplasma genitalium, Ureaplasma urealyticum and Mycoplasma pulmonis. This allowed the identification of large numbers of potentially variable regions, as well as a comparison of the relative recombination potentials of different genomic regions. Different trends are perceptible among mycoplasmas, probably due to different functional and structural constraints. The largest potential for illegitimate recombination in M.pulmonis is found at the vsa locus and its comparison in two different strains reveals numerous changes since divergence. On the other hand, the main M.pneumoniae and M.genitalium adhesins rely on large distant repeats and, hence, homologous recombination for variation. However, the relation between the existence of repeats and antigenic variation is not necessarily straightforward, since repeats of P1 adhesin were found to be anti-correlated with epitopes recognized by patient antibodies. These different strategies have important consequences for the structures of genomes, since large distant repeats correlate well with the major chromosomal rearrangements. Probably to avoid such events, mycoplasmas strongly avoid inverse repeats, in comparison to co-oriented repeats.

Adhesins, Bacterial↗

PubMed2003

REBASE: restriction enzymes and methyltransferases.

REBASE contains comprehensive information about restriction enzymes, DNA methyltransferases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Homing endonucleases are also included. REBASE contains the most complete and up-to-date information about the methylation sensitivity of restriction endonucleases. In addition, there is extensive information about the known and putative restriction-modification (R-M) systems in more than 100 sequenced bacterial and archaeal genomes. The data is available on the web (http://rebase.neb.com/rebase/rebase.html), through ftp (ftp.neb.com) and as monthly updates via email.

DNA Methylation↗

PubMed2005

REBASE--restriction enzymes and DNA methyltransferases.

REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in restriction-modification. It contains both published and unpublished work with information about recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Experimentally characterized homing endonucleases are also included. Additionally, REBASE contains complete and up-to-date information about the methylation sensitivity of restriction endonucleases. An extensive analysis is included of the restriction-modification systems that are predicted to be present in the sequenced bacterial and archaeal genomes from GenBank. The contents of REBASE are available by browsing from the web (http://rebase.neb.com/rebase/rebase.html) and through selected compilations by ftp (ftp.neb.com) and as monthly updates that can be requested via email.

DNA Modification Methylases↗

PubMed2007

REBASE--enzymes and genes for DNA restriction and modification.

REBASE is a comprehensive database of information about restriction enzymes, DNA methyltransferases and related proteins involved in the biological process of restriction-modification. It contains fully referenced information about recognition and cleavage sites, isoschizomers, neoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. Experimentally characterized homing endonucleases are also included. All newly sequenced genomes are analyzed for the presence of putative restriction systems and these data are included within the REBASE. The contents or REBASE may be browsed from the web (http://rebase.neb.com/rebase/rebase.ftp.html) and selected compilations can be downloaded by ftp (ftp.neb.com). Additionally, monthly updates can be requested via email.

DNA Modification Methylases↗

PubMed1996

Isolation and expression of a Xenopus laevis DNA methyltransferase cDNA.

A Xenopus DNA methyltransferase cDNA was isolated from a Xenopus oocyte cDNA library by screening with the mouse DNA methyltransferase cDNA as a probe. The elucidated nucleotide sequence gave a 4,470 nucleotide open reading frame, and the predicted protein was composed of 1,490 amino acid residues, showing high homology to animal DNA methyltransferases, especially in the catalytic domain in the carboxyl-terminal region. The cysteine-rich region and the Lys-Gly repeat which were first found in the mouse sequence were conserved in Xenopus. However, 200 amino acid residues at the amino-terminus of Xenopus DNA methyltransferase were quite different from those of mouse and human, but showed 70% homology with those of chicken. The cloned Xenopus DNA methyltransferase cDNA expressed in COS1 cells showed a significant DNA methyltransferase activity. The size of the translation product of Xenopus DNA methyltransferase cDNA expressed in COS1 cells was identical with that of the endogenous DNA methyltransferase in Xenopus A6 cells and also with the size of newly synthesized DNA methyltransferase in Xenopus oocytes. However, a slightly larger immunoreactive band of about 205 kDa, and a small immunoreactive band of about 100 kDa, which were poorly labeled by short incubation with radiolabeled amino acids, were the main bands in stage I-III and stage IV-VI oocytes, respectively.

Amino Acid Sequence↗

PubMed2003

DNA mismatch repair genes in renal cell carcinoma.

PURPOSE: DNA mismatch repair is one of the correcting mechanisms that preserves genetic stability during replication or chemically induced damage. We hypothesized that genetic instability is due to a defect in mismatch repair genes in renal cell carcinoma. To test this hypothesis mismatch repair genes hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2 were analyzed in renal cell carcinoma cell lines and tissues. We further investigated the mechanisms of inactivation of these genes through CpG methylation pathways. MATERIALS AND METHODS: We analyzed 41 fresh normal and renal cell carcinoma samples for gene and protein expression of various mismatch repair genes (hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2) using reverse transcriptase-polymerase chain reaction and immunohistochemistry techniques. To investigate the mechanisms of inactivation of these genes cultured renal cancer cell lines (A498, Caki-1 and Caki-2) were treated with demethylating agent (5-aza-2'-deoxycytidine), and mismatch repair genes and protein expression were analyzed before and after treatment. RESULTS: hMLH1 and hMSH3 mRNA expression was significantly lower in renal cell carcinoma tissues than in normal tissues. Similarly nuclear positivity of hMSH3 was significantly lower in renal cell carcinoma tissues than in normal tissues. Moreover, at the mRNA and protein levels hMSH3 expression in high grade renal cell carcinomas was significantly lower than in low grade tumors. However, there was no significant difference in hMSH2, hMSH6, hPMS1 or hPMS2 expression in renal cell carcinoma tissues versus normal kidney tissues. In renal cancer cell lines demethylation with 5-aza-2'-deoxycytidine did not affect the expression of hMLH1 and hMSH3 genes. CONCLUSIONS: To our knowledge this is the first comprehensive study demonstrating the down-regulation of mismatch repair genes in renal cell carcinoma. Selective defect in some mismatch repair genes can cause genomic instability and activate the malignant transformation as well as the progression of renal cell carcinoma.

Azacitidine↗

PubMed1990

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes.

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Loss of methylation activates Xist in somatic but not in embryonic cells.

The mouse Xist gene, which is expressed only from the inactive X chromosome, is thought to play a role in the initiation of X inactivation. The 5' end of this gene is fully methylated on the active X chromosome and completely demethylated on the inactive X chromosome, suggesting that DNA methylation may be involved in controlling allele-specific transcription of this gene. To directly investigate the importance of DNA methylation in the control of Xist expression, we have examined its methylation patterns and expression in ES cells and embryos that are deficient in DNA methyltransferase activity. We report here that demethylation of the Xist locus in male mutant embryos induces Xist expression, thus establishing a direct link between demethylation and expression of the Xist gene in the postgastrulation embryo. The transcriptional activity of Xist in undifferentiated ES cells, however, appears to be independent of its methylation status. These results suggest that methylation may only become essential for Xist repression after ES cells have differentiated or after the embryo has undergone gastrulation.

Animals↗

PubMed2001

Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation.

A cytosine DNA methyltransferase containing a chromodomain, Zea methyltransferase2 (Zmet2), was cloned from maize. The sequence of ZMET2 is similar to that of the Arabidopsis chromomethylases CMT1 and CMT3, with C-terminal motifs characteristic of eukaryotic and prokaryotic DNA methyltransferases. We used a reverse genetics approach to determine the function of the Zmet2 gene. Plants homozygous for a Mutator transposable element insertion into motif IX had a 13% reduction in methylated cytosines. DNA gel blot analysis of these plants with methylation-sensitive restriction enzymes and bisulfite sequencing of a 180-bp knob sequence showed reduced methylation only at CpNpG sites. No reductions in methylation were observed at CpG or asymmetric sites in heterozygous or homozygous mutant plants. Our research shows that chromomethylase Zmet2 is required for in vivo methylation of CpNpG sequences.

Amino Acid Sequence↗