Action of bacterial cytidine deaminase on 5,6-dihydrocytidine

Evans, B.; Mitchell, Gordon N.; Wolfenden, Richard

doi:10.1021/bi00674a024

Cited by 25 publications

(14 citation statements)

References 17 publications

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“…(ii) Cytosines are methylated, and then some or all of these are deaminated to give thymines. On the basis of the suggestion that a suspected intermediate in the enzymatic methylation reaction is more than 10 4 times more prone to spontaneous deamination than is cytosine (11,61), we proposed that RIP may be catalyzed by a DNA methyltransferase under special conditions, such as when the methyl group donor S-adenosylmethionine is limiting (40). Supporting this model, recent studies have demonstrated that two bacterial cytosine methyltransferases can catalyze deamination (48,62).…”

Section: Rip8mentioning

confidence: 94%

DNA Methylation Associated with Repeat-Induced Point Mutation inNeurospora crassa

Singer

Marcotte

Selker

1995

Molecular and Cellular Biology

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Repeat-induced point mutation (RIP) is a process that efficiently detects DNA duplications prior to meiosis in Neurospora crassa and peppers them with G:C to A:T mutations. Cytosine methylation is typically associated with sequences affected by RIP, and methylated cytosines are not limited to CpG dinucleotides. We generated and characterized a collection of methylated and unmethylated am RIP alleles to investigate the connection(s) between DNA methylation and mutations by RIP. Alleles of am harboring 84 to 158 mutations in the 2.6-kb region that was duplicated were heavily methylated and triggered de novo methylation when reintroduced into vegetative N. crassa cells. Alleles containing 45 and 56 mutations were methylated in the strains originally isolated but did not become methylated when reintroduced into vegetative cells. This provides the first evidence for de novo methylation in the sexual cycle and for a maintenance methylation system in Neurospora cells. No methylation was detected in am alleles containing 8 and 21 mutations. All mutations in the eight primary alleles studied were either G to A or C to T, with respect to the coding strand of the am gene, suggesting that RIP results in only one type of mutation. We consider possibilities for how DNA methylation is triggered by some sequences altered by RIP.Certain cytosines in the DNA of many animals, plants, and fungi are methylated. The importance of DNA methylation in mice is highlighted by the finding that it is essential for embryogenesis (24). Many clues to the function(s) of methylation are emerging. DNA methylation has been implicated in X-chromosome inactivation (49), genomic imprinting (4, 38), and other processes. Methylation of promoter sequences typically correlates with gene inactivity (1); however, it is unclear to what extent methylation is used by eukaryotic organisms to regulate transcription.Despite progress made in understanding functions of DNA methylation, little is known about the control of methylation in eukaryotes. In mammals, DNA methylation patterns are reorganized during gametogenesis and embryogenesis (28), but the patterns are relatively stable after differentiation of tissues. 5-Methylcytosine (m 5 C) is predominantly located within CpG dinucleotides in animals (50). Riggs (36) and Holliday and Pugh (20) proposed that a ''maintenance methylase'' propagates methylation patterns by acting on hemimethylated 5Ј-CpG-3Ј/GpC dinucleotides in newly replicated DNA. Aspects of this model have been supported, but non-CpG methylation (54, 60) and methylation heterogeneity evidenced by partially methylated sites (56, 63) challenge the model (39). How methylation patterns are first established also remains largely a mystery, although results of recent studies in mammals suggest that binding of the transcription factor Sp1 can keep regions unmethylated (5, 25).The fungus Neurospora crassa provides an excellent system to study DNA methylation. Most of the genome appears devoid of methylation; however, several densely methylated endogenous...

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Section: Rip8mentioning

confidence: 94%

DNA Methylation Associated with Repeat-Induced Point Mutation inNeurospora crassa

Singer

Marcotte

Selker

1995

Molecular and Cellular Biology

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“…The stereochemical configuration at C-4 is drawn by analogy with the observed stereochemistry at C-6 of adenosine deaminase inhibitor (Kati & Wolfenden, 1989b), but remains to be established for cytidine deaminase. Considering the ability of model nucleophiles to catalyze deamination of cytidine by adding to the 5,6-double bond (Shapiro & Klein, 1966Klein, , 1967Notari, 1967;Wechter, 1970;Wechter & Kelly, 1970;Shapiro, 1980), it seemed possible that the action of cytidine deaminase might involve addition of water or an enzyme nucleophile at the 5,6-double bond. That mechanism became less attractive when the enzyme was found to catalyze the hydrolytic deamination of 5,6-dihydrocytidine, in which 5,6-addition of a nucleophile could not occur (Evans et al, 1975). A simpler mechanism of action was suggested by the enzyme's observed sensitivity to the competitive inhibitors shown in Figure 1.…”

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confidence: 99%

Binding of pyrimidin-2-one ribonucleoside by cytidine deaminase as the transition-state analog 3,4-dihydrouridine and contribution of the 4-hydroxyl group to its binding affinity

Frick¹,

Yang²,

Márquez³

et al. 1989

Biochemistry

108

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Cytidine deaminase, purified to homogeneity from constitutive mutants of Escherichia coli, was found to bind the competitive inhibitors pyrimidin-2-one ribonucleoside (apparent Ki = 3.6 x 10(-7) M) and 5-fluoropyrimidin-2-one ribonucleoside (apparent Ki = 3.5 x 10(-8) M). Enzyme binding resulted in a change of the lambda max of pyrimidin-2-one ribonucleoside from 303 nm for the free species to 239 nm for the bound species. The value for the bound species was identical with that of an oxygen adduct formed by combination of hydroxide ion with 1,3-dimethyl-2-oxopyrimidinium (239 nm), but lower than that of a sulfur adduct formed by combination of the thiolate anion of N-acetylcysteamine with 1,3-dimethyl-2-oxopyrimidinium (259 nm). The results suggest that pyrimidin-2-one ribonucleoside is bound by cytidine deaminase as an oxygen adduct, probably the covalent hydrate 3,4-dihydrouridine, rather than intact or as an adduct involving a thiol group of the enzyme. In dilute solution at 25 degrees C, the equilibrium constant for formation of a single diastereomer of 3,4-dihydrouridine from pyrimidin-2-one ribonucleoside was estimated as approximately 4.7 x 10(-6), from equilibria of dissociation of water, protonation of 1-methylpyrimidin-2-one, and combination of the 1,3-dimethylpyrimidinium cation with the hydroxide ion.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…The details of catalytic function of CDA may yield essential clues for conversion of alcohols and olefins to amines as well as in assisting asymmetric synthesis providing important alternative to conventional chemical technologies. The experimental efforts, aimed at [8][9][10]. The role of the active site residues seems to be well recognized, and the reaction energetics are supported by evidence from accurate kinetics and site-directed mutagenesis experiments [11,12].…”

Section: Introductionmentioning

confidence: 99%