N4-methylcytosine as a minor base in bacterial DNA.

Journal of Bacteriology (Impact Factor: 2.69). 04/1987; 169(3):939-43.
Source: PubMed

ABSTRACT The DNA base composition, including the minor base content, of 26 strains of bacteria was determined. The studied bacteria are sources of widely used restriction endonucleases. Approximately 35% of the bacterial DNAs contained N4-methylcytosine, about 60% contained 5-methylcytosine, and about 90% had N6-methyladenine.

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    ABSTRACT: GC-rich regions of the DNA are of special interest, as they generally occur within transcribed or control regions of the genome. However, the high GC-content makes these sequences prone to the formation of hairpin structures, which may persist even at the relatively high temperature of the extension step in PCR protocols, making their amplification difficult in many instances. Conversion to a different nucleotidic alphabet, resulting in thermodynamically weaker versions of the G:C base pair, may provide a resolution of this problem. The 7-deaza analogue of the guanine base has been tried in this context; however, it appears that the c7G:C base pair, while less stable than the canonical G:C pair, is still too strong, so that problems due to hairpin formation persist. N4-alkylated cytosines form specific, but much weaker base pairs with guanines, and may represent a more effective solution to the problem, provided not only that they are stably inserted into the newly formed DNA during the extension step, but also correctly read in subsequent extensions. We have examined the dCTP analogue, N4methyl-dCTP, for its ability to sustain a PCR, both with HotStart Taq DNA polymerase and with Pfu exo− DNA polymerase, amplifying a 200-bp amplicon within the pUC18 sequence. The Taq enzyme produced the expected product with the nucleotide complement dATP/dGTP/dTTP/N4methyl-dCTP, albeit in reduced yield, compared to the yield obtained with the use of dCTP or of dCTP/N4methyl-dCTP mixtures. A slowdown thermal protocol (Frey et al., 2008, Nature Protocols, 3, 1312–1318), using successively lower hybridization temperatures and slow temperature ramps, proved beneficial in giving a high yield of the desired amplicon, with the expected size and the correct nucleotide sequence. The all-N4-methylC amplicon showed a T m reduced by 11 °C, compared to the amplicon obtained with the canonical set of nucleotides, while amplifications performed with mixtures of dCTP and N4methyl-dCTP gave products of the expected size, showing intermediate melting temperatures (see Figure), all of which attests to the lower thermal stability of the N4-methylC:G pair, compared to the G:C base pair. In contrast, the amplicon synthesized with the dATP/c7dGTP/dTTP/dCTP nucleotide set showed a Tm reduction of only 5°C. The Pfu exo−enzyme was less capable of sustaining PCR with the N4-methyl nucleotide, providing a small yield of 200-bp amplicon only in the presence of higher concentrations of N4-methyl-dCTP.
    Journal of biomolecular Structure & Dynamics 01/2013; 31. DOI:10.1080/07391102.2013.786366 · 2.98 Impact Factor
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    ABSTRACT: Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.
    Nucleic Acids Research 10/2013; DOI:10.1093/nar/gkt1039 · 8.81 Impact Factor
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    ABSTRACT: Methylation of cytosine is a common biological process both in prokaryotic and eukaryotic cells. In addition to 5–methylcytosine (5mC), some bacterial species contain in their genome N4–methylcytosine (N4mC). Methylation at C5 has been shown to enhance the formation of pyrimidine dimeric photoproducts but nothing is known of the effect of N4 methylation on UV-induced DNA damage. In the present work, we compared the yield and the nature of bipyrimidine photoproducts induced in a series of trinucleotides exhibiting a TXG sequence where X is either T, C, 5mC or N4mC. HPLC associated to tandem mass spectrometry was used to quantify cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts (64PP) and their Dewar valence isomer. Methylation at position N4 was found to drastically increase the reactivity of C upon exposure to both UVC and UVB and to favor the formation of 64PP. In contrast methylation at C5 increased the yield of CPD at the expense of 64PP. In addition, enhancement of photoreactivity by C5 methylation was much higher in the UVB than in the UVC range. These results show the drastic effect of the methylation site on the photochemistry of cytosine.This article is protected by copyright. All rights reserved.
    Photochemistry and Photobiology 10/2014; 91(1). DOI:10.1111/php.12365 · 2.68 Impact Factor

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