A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy

Frederico, Lisa Ann; Kunkel, Thomas A.; Shaw, Barbara Ramsay

doi:10.1021/bi00462a015

Cited by 477 publications

(428 citation statements)

References 26 publications

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“…In experiments comparable with those reported by earlier investigators (22,23), we extended those measurements to include cytosine residues in single-stranded DNA, using a phagemid in which we sequenced an insert representing the gene of the HIV-1 protease to compare the rates of cytosine-deaminating events with those of other potentially mutagenic reactions in single-stranded DNA (ssDNA).…”

Section: Hiv-1 Proteasementioning

confidence: 82%

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Lewis

Crayle

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5′-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90°C to 200°C and indicated a heat of activation (ΔH ‡ ) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100°C to 25°C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years. spontaneous mutation | heat mutagenesis | cytosine deamination | HIV-1 protease I n the absence of enzymes, most biological reactions take place so slowly that they can be followed conveniently only at elevated temperatures (1). Among those reactions are several that may result in spontaneous mutation, notably the hydrolytic deamination of cytosine and 5-methylcytosine, which generate uracil and thymine, respectively. These deamination reactions have been shown to account for many of the single-site mutations that lead to inherited diseases in humans (2) and for the marked bias of spontaneous mutation toward increased AT content in microorganisms (3, 4). Rates of mutation have long been known to increase with increasing temperature, a phenomenon that Drake has termed "heat mutagenesis" (5).There is widespread (6-8), if not universal (9), agreement that life originated when the earth was warmer-perhaps much warmer-than it is today. A recent isotopic analysis of carbon inclusions in zircons from the Jack Hills in Western Australia indicates that life may have emerged as early as 4.1 billion years ago (10), shortly after water first appeared at the surface in liquid form (11). Several present-day organisms thrive at temperatures near the boiling point of water. Thus, Ignisphera aggregans (12) and Pyrococcus horikoshii (13) exhibit optimal growth temperature of 92°and 98°C, respectively, whereas another organism isolated from a hydrothermal vent has be...

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Section: Hiv-1 Proteasementioning

confidence: 82%

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Lewis

Crayle

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…This can be associated with the presence of CpG methylation, which can act as a repressive epigenetic mark in some contexts (34). Cytosine methylation makes the base susceptible to deamination, resulting in C-to-T transitions (35,36). Thus, CpG frequency can be used as an indication of the extent of methylation.…”

Section: Resultsmentioning

confidence: 99%

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano

Wasik

Gurtowski

Zhou

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

100

138

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The free-living flatworm, Macrostomum lignano has an impressive regenerative capacity. Following injury, it can regenerate almost an entirely new organism because of the presence of an abundant somatic stem cell population, the neoblasts. This set of unique properties makes many flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cellfate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of M. lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ∼75% of its sequence being comprised of simple repeats and transposon sequences. This has made high-quality assembly from Illumina reads alone impossible (N50 = 222 bp). We therefore generated 130× coverage by long sequencing reads from the Pacific Biosciences platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene-expression patterns during regeneration, examining pathways important to stem cell function.F latworms belong to the superphylum Lophotrochozoa, a vast assembly of protostome invertebrates (1, 2) (Fig. 1A). The evolutionary relationships within this clade are poorly resolved and the specific position of flatworms is currently debated (3, 4). Flatworms have attracted scientific attention for centuries because of their astonishing regenerative capabilities (5, 6), as well as their ability to "degrow" in a controlled way when starved (7). As far back as the early 1900s, Thomas Morgan recognized the potential of flatworms and conducted a number of fascinating regeneration experiments on planarian flatworms before his focus shifted to Drosophila genetics (8).Macrostomum lignano is (Fig. 1B), a free-living, regenerating flatworm isolated from the coast of the Mediterranean Sea. M. lignano is an obligatorily cross-fertilizing simultaneous hermaphrodite (9) that belongs to Macrostomorpha, whereas the other often-studied freeliving flatworms and human parasitic flatworms all belong to clades that are potentially more derived (less ancestral) in comparison with Macrostomorpha (2) (Fig. 1C).Many flatworms can regenerate nearly their entire body or amputated organs. This regenerative capacity is thought to be attributable to the presence of somatic stem cells, termed neoblasts (10, 11). In Schmidtea mediterranea (planarian flatworm), even a single transplanted neoblast has the ability to rescue, regenerate, and change the genotype of a fatally irradiated worm (12). M. lignano can regenerate every tissue, with the exception of the head region containing the brain (13,14).Neoblasts in M. lignano ( Fig. 1 D and E), in contrast to most vertebrate somatic stem cells, are plentiful, making up about ...

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“…27 The non-transcribed strand is transiently single-stranded, in contrast to the transcribed strand that may largely be protected by the R-loop. Spontaneous (5-methyl)cytosine deamination in singlestrand DNA is increased about 100-fold, 28 which provides a mechanistic explanation for this TAP pathway. Later work has revealed that also the frequency of spontaneous nucleotide transversions at the nontranscribed strand is increased.…”

Section: Transcription-associated Point Mutagenesis (Tap)mentioning

confidence: 99%

Transcription and replication: Far relatives make uneasy bedfellows

Hendriks¹,

Jansen²,

Mullenders³

et al. 2010

Cell Cycle

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A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy

Cited by 477 publications

References 26 publications

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history

Genome and transcriptome of the regeneration-competent flatworm, Macrostomum lignano

Transcription and replication: Far relatives make uneasy bedfellows

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