Why is Cytosine Arabinoside Administered Continuous
Cytosine
August W. Holldorf , in Methods of Enzymatic Analysis (Second Edition), Volume 4, 1974
Publisher Summary
This chapter provides an overview of cytosine that is a precursor of cytosine nucleotides and, therefore, of ribo- and deoxyribonucleic acids. In most organisms, the free form is not an intermediate in the biosynthesis or catabolism of nucleotides and nucleic acids. Consequently, so far the free form has either not been found or only detected in very small amounts in biological material. Cytosine is, however, obtained in the chemical or enzymatic degradation of nucleic acids. Identification and determination is usually based on the UV absorption spectrum. Cytosine is deaminated to uracil by cytosine deaminase. That reaction is the basis for the specific determination of cytosine. The reaction is accompanied by a decrease of extinction at 280 nm 2. The decrease in extinction is proportional to the amount of cytosine reacting. The equilibrium of reaction lies completely on the side of uracil formation. The enzyme from yeast has a pH optimum between 7.0 and 7.4; it requires no cofactors. Cytosine deaminase also reacts with 5-methylcytosine, which occurs in small amounts in several nucleic acids, as well as with cytosine derivatives substituted with halogens in the 5 position.
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DNA Methylation and Hydroxymethylation in Cancer
Fazila Asmar , ... Kirsten Grønbæk , in Epigenetic Cancer Therapy, 2015
| CpG islands |
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| CpG shores |
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| CpG shelves |
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| CpG canyon |
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| CpG ocean |
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| Gene body |
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| Gene desert |
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DEOXYRIBONUCLEOSIDE METABOLISM
J. Frank Henderson , A.R.P. Paterson , in Nucleotide Metabolism, 1973
2 Cytosine Arabinoside
Cytosine arabinoside, an analog of deoxycytidine, is a substrate for deoxycytidine kinase and is a competitive inhibitor of the phosphorylation of the natural substrate; the Michaelis constant for the calf thymus enzyme is 10 times higher than that of deoxycytidine ( 35 ). Cytosine arabinoside inhibits the growth of a variety of experimental neoplasms and has an important use in the chemotherapy of human cancer. The lethal effects of the analog toward malignant and other cells are generally attributed to inhibition of DNA synthesis. Cytosine arabinoside is extensively anabolized in cells; the mono-, di-, and triphosphate derivatives are formed and some incorporation into internal nucleotides in DNA occurs. The drug-induced inhibition of DNA synthesis has been attributed to the competitive inhibition of DNA polymerase by cytosine arabinoside triphosphate ( 41 , 42 ). A necessary first step in this mechanism of action would be the kinase-catalyzed phosphorylation of cytosine arabinoside; the limited activity of deoxycytidine kinase in resistant mouse lymphoma cells is consistent with this mechanism ( 43 ). The ability of deoxycytidine to protect cells against the inhibitory effects of cytosine arabinoside is presumably due in some measure to the competition between deoxycytidine and the arabinoside for the catalytic site of deoxycytidine kinase. Cytosine arabinoside is deaminated by cytidine deaminase to form the pharmacologically inactive uracil arabinoside.
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The Functional Genome
Timothy E. Reddy , in Genomic and Precision Medicine (Third Edition), 2017
The Establishment and Maintenance of 5mC
Cytosines are methylated by the DNA methyltransferase (DNMT) family of enzymes [4]. The DNMTs transfer a methyl group from S-adenosyl-l-methionine (SAM) to the 5' carbon of cytosine residues in the genome [5]. There are two known subfamilies of DNMTs in the human genome with different properties. The DNMT3 subfamily consists of DNMT3A and DNMT3B and is responsible for methylating cytosine de novo [6]. Once methylation is established on one strand of the double helix, the DNMT1 subfamily, made up solely of DNMT1, is thought to methylate the corresponding cytosine on the opposite strand [7,8]. In that capacity, DNMT1 is sometimes considered to be a post-replication maintenance DNMT. DNMT1 also has the ability to methylate DNA de novo in certain regions of the genome [9], suggesting that the division of labor between DNMT1, DNMT3A, and DNMT3B is neither strict nor simple [4]. The formerly-named DNMT2 enzyme was found to methylate transfer RNA (tRNA) and not DNA [10,11]. For that reason, DNMT2 has since been renamed the tRNA aspartic acid methyltransferase 1 (TRDMT1). RNA methylation by TRDMT1 may be related to a more extensive RNA-based epigenome that will not be discussed in this chapter.
The mechanism by which cytosine methylation is erased remains a highly active area of research. Two predominant mechanisms have emerged [12,13]. The passive model of cytosine demethylation relies on a failure to methylate newly synthesized DNA during mitosis, perhaps involving the inhibition of DNMT1. If the cytosine methylation is systematically not established at a given genomic location after DNA replication, then the number of chromosomes with cytosine methylation at that location in the two daughter cells will be reduced by half. With subsequent rounds of mitosis and new DNA synthesis, the vast majority of daughter cells will eventually have nonmethylated cytosines at that location [13]. The appeal of the passive model in humans is that it does not depend on the presence of an active cytosine demethylation enzyme or pathway that has long eluded discovery. The requirement for mitosis, however, restricts passive demethylation to dividing cells and does not explain, for example, the rapid loss of methylation that occurs on the paternal genome after a sperm cell fertilizes an egg but before the first rounds of mitotic cell division [14,15].
More recently, increasing evidence also supports a second, active mechanism of demethylation that does not depend on cell division. The base-excision-repair hypothesis states that methylated cytosines are excised from the genome and replaced by nonmodified cytosines. Meanwhile, the enzymatic demethylation hypothesis states that the methyl group on 5mC is directly removed from the cytosine by a demethylase enzyme. Demethylation by base-excision repair has been demonstrated in plants, but has not been shown to occur in the human genome [16]. Enzymatic removal of the methyl group from 5mC by a single enzyme is thought to be energetically unlikely. Instead, the possibility of a demethylation pathway that relies on successive modifications of the 5mC by several enzymes has recently gained attention. The ten-eleven translocation (TET) enzymes convert 5mC into 5hmC, which may be the first step in a chain of further modifications that ultimately result in unmodified cytosine [16–18]. Human enzymes that convert 5hmC or 5hmC derivatives to cytosine, however, have not yet been found.
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Structure and Function of Human DNA Methyltransferases
R.Z. Jurkowska , A. Jeltsch , in Reference Module in Biomedical Sciences, 2014
Base Flipping and General Catalytic Mechanism of Cytosine-C5 MTases
Cytosine-C5 DNA MTases catalyze the transfer of the methyl group from a cofactor molecule S-adenosyl- l-methionine (AdoMet) to the C5 position of cytosine residues in DNA (Figure 1). In this reaction, the 5-methylcytosine is created and the S-adenosyl-l-homocysteine (AdoHcy) is released from the enzyme. The transfer of the activated methyl group from AdoMet to the C5 position of the cytosine requires a close contact between the enzyme's active site and the substrate base. Such proximity is not possible while the base is located in the DNA double helix; therefore DNA MTases flip their target base out of the DNA during catalysis and bury it into a hydrophobic pocket of their active center. The base flipping mechanism was first discovered in 1994 for the bacterial DNA C5 MTase M.HhaI (Klimasauskas et al., 1994). Later, it became clear that it is common to all DNA MTases (Cheng and Roberts, 2001; Jeltsch, 2002) and other enzymes interacting with DNA (Roberts and Cheng, 1998). Flipping of the cytosine base could also be observed in the crystal structure of human DNA nucleotide methyltransferase 1 (Dnmt1) that has been solved recently (see below) (Song et al., 2012).
Chemically, the methylation of the C5 position of cytosine is not an easy task, because cytosine is an electron-poor aromatic system and the C5 position is not intrinsically reactive, such that it will not attack the activated methylsulfonium group of AdoMet spontaneously. Therefore, a key step in the catalysis is the nucleophilic attack of a cysteine residue from the active center of the enzyme on the sixth position of the target cytosine, leading to the formation of a covalent bond between the enzyme and the substrate base (Figure 3). Thereby, the negative charge density at the C5 atom of the cytosine increases, such that it can attack the methyl group of the cofactor. It has been proposed that the nucleophilic attack of the cysteine might be facilitated by a transient protonation of the cytosine at its endocyclic N3 nitrogen atom by a conserved glutamate residue. The catalytic cysteine and the glutamic acid are located in two highly conserved amino acid (aa) motifs, the PCQ and ENV, respectively. The addition of the methyl group to the base is followed by a deprotonation of the C5 atom, catalyzed by a so far unknown proton acceptor, which resolves the covalent bond between the enzyme and the base in an elimination reaction and re-establishes aromaticity (Cheng and Roberts, 2001; Jeltsch, 2002).
Figure 3. Catalytic mechanism of cytosine-C5 DNA MTases. The catalytic glutamate residue from ENV motif (motif IV) is colored brown, the catalytic cysteine residue from PCQ motif (motif IV) is colored green, AdoMet is blue, and the methyl group is shown in red. The base for final proton abstraction (shown in orange) is not identified; it may be an amino acid residue or a water molecule.
Adapted from Jeltsch, A., 2002. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem 3, 274–293.Read full chapter
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Epigenetics: Analysis of Cytosine Modifications at Single Base Resolution
Malwina Prater , Russell S. Hamilton , in Encyclopedia of Bioinformatics and Computational Biology, 2019
Long-Read Sequencing
Cytosines undergoing modifications are often located in clusters (e.g., CpG islands) which are therefore repetitive in nature. Short-read based methods are unable to uniquely map short reads to repetitive regions, resulting in the under representation of these important regions. Long-read based methods such as Pacific Biosciences single-molecule real-time sequencing (SMRT) and Oxford Nanopore do not suffer from this limitation as the reads can be 10's or 100's of kilobases in length and can therefore span and uniquely map to these CpG dense regions. A further key advantage is the ability to directly assess the methylation status of cytosines without the need for treatments such as bisulfite, or enzymatic modification. SMRT sequencing measures the incorporation of nucleotides in real time and is able to distinguish between each of the modification states of cytosine and adenine ( Fang et al., 2012; Suzuki et al., 2015), while Nanopore technology detects DNA bases, including modified cytosines, by measuring electrostatic charge as a DNA strand passes through a protein nanopore (Rand et al., 2017; Simpson et al., 2017). The challenge now is to develop new bioinformatics methods for long-read sequencing such as high accuracy base calling of methylated bases
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RNA Modification
Vahid Khoddami , ... Bradley R. Cairns , in Methods in Enzymology, 2015
Abstract
RNA cytosine methyltransferases (m 5C-RMTs) constitute an important class of RNA-modifying enzymes, methylating specific cytosines within particular RNA targets in both coding and noncoding RNAs. Almost all organisms express at least one m5C-RMT, and vertebrates often express different types or variants of m5C-RMTs in different cell types. Deletion or mutation of particular m5C-RMTs is connected to severe pathological manifestations ranging from developmental defects to infertility and mental retardation. Some m5C-RMTs show spatiotemporal patterns of expression and activity requiring careful experimental design for their analysis in order to capture their context-dependent targets. An essential step for understanding the functions of both the enzymes and the modified cytosines is defining the one-to-one connection between particular m5C-RMTs and their target cytosines. Recent technological and methodological advances have provided researchers with new tools to comprehensively explore RNA cytosine methylation and methyltransferases. Here, we describe three complementary approaches applicable for both discovery and validation of candidate target sites of specific m5C-RMTs.
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Methods for Nonradioactive Labeling of Nucleic Acids
Christoph Kessler , in Nonisotopic Probing, Blotting, and Sequencing (Second Edition), 1995
4 Derivatization with Mercury
Cytosine and uracil nucleotides are readily mercurated by heating at 37 to 50° C with mercuric acetate in buffered aqueous solutions (pH 5.0-8.0) ( Dale et al., 1975). Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA, whereas only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of nucleic acid mercuration is influenced by ionic strength; the lower the ionic strength, the faster the reaction. The mild reaction conditions give minimal strand-breakage and do not produce pyrimidine hydrates.
The position of mercuration of cytosine and uracil is C-5 (Bergstrom and Ruth, 1977); in uracil, this modification mimics the methyl group of thymine. Although sufficiently stable to permit biochemical studies, the mercury–carbon bond is extremely sensitive to cleavage by electrophiles and reducing agents. Treatment of mercurated polynucleotides with I2, N- bromosuccinimide has been found to generate the corresponding iodinated and brominated nucleic acids rapidly. Therefore, the mercurated, iodinated, or brominated nucleic acids can be either detected directly or further substituted with activated linker arms carrying a detectable hapten such as biotin (Langer et al., 1981; Hopman et al., 1986a, 1986b; Keller et al., 1988).
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m5C Cytosine methyltransferase
Harry Morrison , in Enzyme Active Sites and their Reaction Mechanisms, 2021
12.1 m5C Cytosine methyltransferase
m5C Cytosine methyltransferase (EC 2.1.1.37; C-5 cytosine-specific DNA methylase; m 5C-MTase) is one of several enzymes that methylate cytosine or adenine in DNA. It specifically methylates the C-5 carbon of cytosine; the others methylate the N4 position of cytosine and the N6 position of adenine. m5C is the most common methylase in mammals and is labeled DNMT1 (DNA methyltransferase 1). However, much of the structural and mechanistic information about this class of enzymes is derived from studies on a bacterial analog, M.HhaI, derived from Haemophilus haemolyticus. There is considerable conservation of the AA's at the active site between these two (and with m5C-MTases in general) and the mechanisms for the two methylases are quite similar. M.HhaI recognizes the 5′-GCGC-3′ sequence and methylates the underlined cytosine.
The requisite cofactor for this reaction is a ubiquitous methylating agent, S-adenosyl-l-methionine (AdoMet). (AdoMet is formed by the displacement of inorganic phosphate from ATP by the sulfur atom of methionine). The demethylation of AdoMet leads to S-adenosyl-l-homocysteine (AdoHcy; see Figs. 12.1 and 12.2).
Figure 12.1. Structures of AdoMet and AdoHcy.
Figure 12.2. Overall reaction of m5C-MTase.
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Neuroepigenetics and Mental Illness
Elizabeth J. Radford , in Progress in Molecular Biology and Translational Science, 2018
2.2 DNA Hydroxymethylation
Cytosine hydroxymethylation (5hmC) is produced by the oxidation of 5-methyl-cytosine, a reaction catalyzed by the TET family of enzymes. 57,58 5hmC is not recognized by DNMT1 nor most methyl-binding domain containing proteins, and is thought to functionally resemble unmodified cytosine to the cellular apparatus. 58 All three mammalian TET enzymes can convert 5mC to 5hmC, further TET activity can convert 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which are subsequently recognized and excised by thymine DNA glycosylase (TDG) to be replaced with unmethylated cytosine. 59,60 This is thought to be the mechanism through which regions are actively demethylated in mammals. 5hmC is relatively abundant in mouse embryonic stem cells, ESCs, the early embryo and in adult brain. 57,61–63 Hydroxymethylation of pluripotency genes in ESCs is important in the maintenance of pluripotency. 64 However, the full role of hydroxymethylation in the brain, as well as other tissues, remains to be elucidated.
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