DNA modifications

The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cover Caption: Pictured is a mouse 1-cell embryo double-stained with an anti-5mC antibody (red) and an anti-5fC antibody (green) at the prometaphase during the first mitosis of life. The chromosomes inherited from the father have undergone oxidation of 5mC, as shown by 5fC staining, whereas the chromosomes inherited from the mother are rich in 5mC. Both sets of condensing chromosomes form a cartwheel structure. In the paternal cartwheel, a 5mC-rich, oxidation-resistant circle appears to organize the 5fC-rich, radially arranged chromosomes. Image courtesy of Tie-

Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions. In prokaryotes, modified bases appear primarily to be part of an arms race between bacteriophages (and other genomic parasites) and their hosts, although, as in eukaryotes, some modifications have been adapted to convey epigenetic information. The first half of this review catalogs the identification and diversity of DNA modifications found in bacteria and bacteriophages. What is known about the biogenesis, context, and function of these modifications are also described. The second part of the review places these DNA modifications in the context of the arms race between bacteria and bacteriophages. It focuses particularly on the defense and counter-defense strategies that turn on direct recognition of the presence of a modified base. Where modification has been shown to affect other DNA transactions, such as expression and chromosome segregation, that is summarized, with reference to recent reviews.

Prolonged exposure to aristolochic acid (AA) contaminated slimming drugs and food is believed to be associated with the development of endemic nephropathy in Belgian women and in farmers living alongside the Danube River. Decades of research has revealed the pathophysiology of carcinogenesis of AA, and the molecular mechanisms underlying renal interstitial fibrosis remain unclear. We hypothesized that RNA modification may have contributed to the observed toxicity of AA. Thus, a highly sensitive and selective ultra-high performance liquid chromatography-coupled tandem mass spectrometric method was developed to quantify RNA-AA adducts in target and nontarget organs of AA-dosed rats. The results revealed, for the first time, that AA forms RNA adducts in vitro and in vivo. Comparative studies on DNA revealed that RNA is modified by AA at frequencies approximately 6-fold higher than that of DNA in both kidney and liver tissue in AA-dosed rats. Results also demonstrated that guanosine is modified by AA at frequencies significantly higher than that of adenosine, 2-deoxyadenosine, and 2-deoxyguanosine in both organs of the AA-dosed. This finding suggests that guanosine is a major target for AA and that guanosine adducts of AA might be critical lesions in the pathophysiology of AA-induced toxicity. It is anticipated that the results of our study may open up a new area of investigating the nephrotoxicity and/or carcinogenicity by quantifying RNA adducts using the UPLC-MS/MS technique of high sensitivity and selectivity.

Under collisional activation condition, it is well known that dissociations of multideprotonated oligonucleotides involve an initial loss of nucleic base (formation of [M−nH-B i ] n−) yielding consecutively the complementary (a i-B) and w j product ions. The loss of thymine is thermodynamically unfavored owing to its low proton affinity. 6 mer DNA anions were activated in SORI-CID and by EDD. The EDD spectra are significantly different from SORI-CID. All EDD spectra showed singly charged w 5 − with good abundance, while this ion was not detected in SORI-CID spectra. In EDD spectra loss of thymine is easily detected, surprisingly doubly charged fragment ions were also detected, these ions can be produced directly from precursor ion [M−2H] 2−. MS 3 and double resonance experiments have been realized to find the origin of these doubly charged and [a i-T] − ions in EDD spectra of d(T 2 AT 3) and (T 2 CT 3). It has been demonstrated that these doubly charged ions are produced by electron induced dissociation. The combination of double resonance and EDD/SORI-CID MS 3 experiments allows decoupling the EID from the EDD processes.

Background Arthrofibrosis, occurring in 3%-4% of patients following total knee arthroplasty (TKA), is a challenging condition for which there is no defined cause. The hypothesis for this study was that disregulated production of reactive oxygen species (ROS) and nitrogen species (RNS) mediates matrix protein and DNA modifications, which result in excessive fibroblastic proliferation. Results We found increased numbers of macrophages and lymphocytes, along with elevated amounts of myeloperoxidase (MPO) in arthrofibrotic tissues when compared to control tissues. MPO expression, an enzyme that generates ROS/RNS, is usually limited to neutrophils and some macrophages, but was found by immunohistochemistry to be expressed in both macrophages and fibroblasts in arthrofibrotic tissue. As direct measurement of ROS/RNS is not feasible, products including DNA hydroxylation (8-OHdG), and protein nitrosylation (nitrotyrosine) were measured by immunohistochemistry. Quantification of the staining...

Double-headed nucleoside monomers have immense applications for studying secondary nucleic acid structures. They are also well-known as antimicrobial agents. This review article accounts for the synthetic methodologies and the biological applications of double-headed nucleosides.

Double-headed nucleoside monomers have immense applications for studying secondary nucleic acid structures. They are also well-known as antimicrobial agents. This review article accounts for the synthetic methodologies and the biological applications of double-headed nucleosides.

The text and figures in Chapter 2, in part or in full, are a reprint of the material as it appears in Clin. Chim. Acta 2012, X, XX-XX and the supporting information therein. The co-author, Dr. Yinsheng Wang, listed in that publication directed and supervised the research, which forms the basis of this chapter. The text and figures in Chapter 4, in part or in full, are a reprint of the material as it appears in Biochemistry 2012, 51, 6458-6462. The co-author, Dr. Yinsheng Wang, listed in that publication directed and supervised the research, which forms the basis of this chapter. v DEDICATION This dissertation is dedicated to my parents, Derrick and Wanda, and to my sister, Erricka.

Alkylation and oxidation constitute major routes of DNA damage induced by endogenous and exogenous genotoxic agents. Understanding the biological consequences of DNA lesions often necessitates the availability of oligodeoxyribonucleotide (ODN) substrates harboring these lesions, and sensitive and robust methods for validating the identities of these ODNs. Tandem mass spectrometry is well suited for meeting these latter analytical needs. In the present study, we evaluated how the incorporation of an ethyl group to different positions (i.e., O 2 , N3 and O 4) of thymine and the oxidation of its 5-methyl carbon impact collisionally activated dissociation (CAD) pathways of electrospray-produced deprotonated ions of ODNs harboring these thymine modifications. Unlike an unmodified thymine, which often manifests poor cleavage of the C3′-O3′ bond, the incorporation of an alkyl group to the O 2 position and, to a much lesser extent, the O 4 position, but not the N3 position of thymine, led to facile cleavage of the C3′-O3′ bond on the 3′ side of the modified thymine. Similar efficient chain cleavage was observed when thymine was oxidized to 5-formyluracil or 5-carboxyluracil, but not 5-hydroxymethyluracil. Additionally, with the support of computational modeling, we revealed that proton affinity and acidity of the modified nucleobases govern the fragmentation of ODNs containing the alkylated and oxidized thymidine derivatives, respectively. These results provided important insights into the effects of thymine modifications on ODN fragmentation.

Nucleic acids are not only a source of life but also a means of observing, understanding, and regulating it. Nucleic acids, DNA and RNA, and their characteristics are discussed in other chapters of the book. This chapter describes the fundamental principles of different methods for nucleic acid sample preparation / nucleic acid extraction, such as columnbased methods using silica membranes and traditional ones without a column purification procedure (commercially available or homemade). Other topics discussed here include comparative analysis of the use of these methods in DNA and RNA extraction from a variety of biological and clinical samples, as well as the relationship between the type of sample, the method used and the quality and amount of extracted DNA or RNA. Finally, the chapter outlines the application of nucleic acids in the diagnosis of various diseases, in scientific research, and bird sex determination by downstream applications such as restriction enzyme analysis, polymerase chain reactions (PCR, reverse transcription-PCR, real-time PCR), and different sequencing methods (Sanger, cycling sequencing, and next-generation sequencing).

We report here the fragmentation mechanism for five 2-acylamino-1,4-naphthoquinone derivatives using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analyses were performed on a lowresolution, triple-quadrupole mass spectrometer. Fragmentation pathways for protonated molecular derivatives 2-acylamino-1,4-naphthoquinone [M + H] + are proposed on the basis of theoretical calculations. There is evidence that the nitrogen atom is the protonation site, based on the nucleophilic atomic indices.

OF THE DISSERTATION ....................................................................... ii DEDICATION ................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................. v TABLE OF CONTENTS ............................................................................................... vii LIST OF FIGURES ....................................................................................................... xiii LIST OF TABLES ......................................................................................................... xix Chapter

We determined the gas-phase acidities (ΔH acid ) of four deoxyribonucleosides, ie, 2′-deoxyadenosine (dA), 2′-deoxyguanosine (dG), 2′-deoxycytidine (dC), and 2′-deoxythymidine (dT) by applying the extended kinetic method. The negatively charged proton-bound hetero-dimeric ...

The mass-analysed ion kinetic energy spectrum of isomeric dinucleotides having dTpdU and dUpdT structure is characterized by the preferred elimination of the pyrimidine base linked to the 5' end deoxyribose unit. Similar results have been obtained with differently substituted dimers such as dCpdT and DMTdTdS)dU. The observed selectivity in the glycosidic bond dissociation provides a straightforward criterion for the sequencing of the sampled species.

The primary structure of 3-imino[60]fulleryl-3-deoxythymidine ions is studied using mass spectrometry both in the positive and negative modes. Interaction between the subunits is discussed using collisioninduced dissociation (CID) spectra. Collisional activation with argon of the sodiated cations leads to the cleavage of the glycosidic bond and the transfer of a radical hydrogen from the deoxyribose to the thymine. The sodiated thymine is the only fragment observed for low collision energies in the positive mode. In the negative mode, two different ionization mechanisms take place, reduction and deprotonation in the presence of triethylamine. The 2.7 eV electron affinity of C 60 and its huge cross section compared to the small cross section and predicted 0.44 eV electron affinity of the thymidine subunit most likely localize the radical electron on the fullerene. On the other hand, deprotonation of the 3-azido-3-deoxythymidine (AZT) is known to occur in N-3, the most acidic site of the nucleobase. Consequently, deprotonation causes the negative charge to be initially localized on the thymine. Both types of parent anions give the radical anion C 60 ž− as fragment. The other fragments detected are the dehydrogenated 3-imino[60]fulleryl-3deoxyribose anion, C 60 NH 2 − , C 60 N − and C 60 H −. Since in negative ion mass spectrometry all fragments include the [60]fullerene unit, this suggests that the fragmentation is driven by the electron affinity of the [60]fullerene, likely responsible for a charge transfer between the deprotonated thymine and the C 60 .

Under collisional activation condition, it is well known that dissociations of multideprotonated oligonucleotides involve an initial loss of nucleic base (formation of [M−nH-B i ] n− ) yielding consecutively the complementary (a i -B) and w j product ions. The loss of thymine is thermodynamically unfavored owing to its low proton affinity. 6 mer DNA anions were activated in SORI-CID and by EDD. The EDD spectra are significantly different from SORI-CID. All EDD spectra showed singly charged w 5 − with good abundance, while this ion was not detected in SORI-CID spectra. In EDD spectra loss of thymine is easily detected, surprisingly doubly charged fragment ions were also detected, these ions can be produced directly from precursor ion [M−2H] 2− . MS 3 and double resonance experiments have been realized to find the origin of these doubly charged and [a i -T] − ions in EDD spectra of d(T 2 AT 3 ) and (T 2 CT 3 ). It has been demonstrated that these doubly charged ions are produced by electron induced dissociation. The combination of double resonance and EDD/SORI-CID MS 3 experiments allows decoupling the EID from the EDD processes.

The discovery and study of toxin-antitoxin (TA) systems helps us advance our understanding of the strategies prokaryotes employ to regulate cellular processes related to the general stress response, such as defense against phages, growth control, biofilm formation, persistence, and programmed cell death. Here we identify and characterize a TA system found in various bacteria, including the global pathogen Mycobacterium tuberculosis. The toxin of the system (DarT) is a domain of unknown function (DUF) 4433, and the antitoxin (DarG) a macrodomain protein. We demonstrate that DarT is an enzyme that specifically modifies thymidines on single-stranded DNA in a sequence-specific manner by a nucleotide-type modification called ADP-ribosylation. We also show that this modification can be removed by DarG. Our results provide an example of reversible DNA ADP-ribosylation, and we anticipate potential therapeutic benefits by targeting this enzyme-enzyme TA system in bacterial pathogens such as M. tuberculosis.

The primary structure of 3'-imino[60]fulleryl-3'-deoxythymidine ions is studied using mass spectrometry both in the positive and negative modes. Interaction between the subunits is discussed using collision-induced dissociation (CID) spectra. Collisional activation with argon of the sodiated cations leads to the cleavage of the glycosidic bond and the transfer of a radical hydrogen from the deoxyribose to the thymine. The sodiated thymine is the only fragment observed for low collision energies in the positive mode. In the negative mode, two different ionization mechanisms take place, reduction and deprotonation in the presence of triethylamine. The 2.7 eV electron affinity of C60 and its huge cross section compared to the small cross section and predicted 0.44 eV electron affinity of the thymidine subunit most likely localize the radical electron on the fullerene. On the other hand, deprotonation of the 3'-azido-3'-deoxythymidine (AZT) is known to occur in N-3, ...

Shift reagents in ion mobility spectrometry: the effect of the number of interaction sites, size and interaction energies on the mobilities of valinol and ethanolamine SHORT TITLE: Shift reagents in IMS: effect of the number of interaction sites Abstract INTRODUCTION Overlapping peaks interfere in ion mobility spectrometry (IMS) but they are separated introducing mobility shift reagents (SR) in the buffer gas forming adducts with different collision cross-sections (size). IMS separations using SR depend on the ion mobility shifts which are governed by adduct's size and interaction energies (stabilities). METHODS Mobility shifts of valinol and ethanolamine ions were measured by electrospray-ionization ion mobility-mass spectrometry (MS). Methyl-chloro propionate (M) was used as SR; 2-butanol (B) and nitrobenzene (N) were used for comparison. Density functional theory was used for calculations. RESULTS B produced the smallest mobility shifts due to its small size. M and N have two strong interaction sites (oxygen atoms) and similar molecular mass and they should produce similar shifts. For both ethanolamine and valinol ions, stabilities were larger for N adducts than those of M. With ethanolamine, M produced a 68% shift, large compared to that using N, 61%, because M has a third weak interaction site on the chlorine atom and, therefore, M has more interaction possibilities than N. This third site overrode the oxygen atoms' interaction energy that favored the adduction of ethanolamine with N over that with M. On the contrary, with valinol mobility shifts were larger with N than with M (21 vs. 18%) because interaction energy favored even more adduction of valinol with N than with M; this is, the interaction energy difference between adducts of valinol with M and N was larger than that between those adducts with ethanolamine, and the third M interaction could not override this larger difference. CONCLUSIONS Mobility shifts were explained based on the number of SR's interaction sites, size of ions and SR, and SR-ion interaction energies. This is the first time that the number of interaction sites is used to explain mobility shifts in SR-assisted IMS.

"Alkylation and oxidation constitute major routes of DNA damage
induced by endogenous and exogenous genotoxic agents.
Understanding the biological consequences of DNA lesions often
necessitates the availability of oligodeoxyribonucleotide (ODN)
substrates harboring these lesions, and sensitive and robust
methods for validating the identities of these ODNs. Tandem mass
spectrometry is well suited for meeting these latter analytical needs.  In the present study, we evaluated how the incorporation of an ethyl group to different positions (i.e., O2, N3, and O4) of thymine and the oxidation of its 5-methyl carbon impact collisionally activated dissociation (CAD) pathways of electrospray-produced deprotonated ions of ODNs harboring these thymine modifications.  Unlike an unmodified thymine, which often manifests poor cleavage of the C3′–O3′ bond, the incorporation of an alkyl group to the O2 position and, to a much lesser extent, the O4 position, but not the N3 position of thymine, led to facile cleavage of the C3′–O3′ bond on the 3′ side of the modified thymine. Similar efficient chain cleavage was observed when thymine was oxidized to 5-formyluracil or 5-carboxyluracil, but not 5-hydroxymethyluracil.  Additionally, with the support of computational modeling, we revealed that proton affinity and acidity of the modified nucleobases govern the fragmentation of ODNs containing the alkylated and oxidized thymidine derivatives, respectively. These results provided important insights into the effects of thymine modifications on ODN fragmentation."

Modifications to deoxyribonucleic acids (DNA) may arise endogenously or be initiated by exogenous sources. In this dissertation, we focus on utilizing mass spectrometry (MS), liquid chromatography (LC), and density functional theory (DFT)
methods to characterize DNA modifications induced by chemotherapeutic agents, and to assess the roles kinetics and thermodynamics play in the removal of oxidized pyrimidines
by human thymine DNA glycosylase (hTDG).

In part I, we conducted an enzymatic digestion study coupled with detailed MS, tandem MS (MS/MS), and LC-MS/MS analyses of oligodeoxyribonucleotides (ODNs) containing cisplatin adducts (Chapter 2). We identified and characterized several digestion products for ODNs containing a 1,2-GpG, 1,2-ApG or 1,3-GpXpG intrastrand cross-link. These results built a solid foundation for the future LC-MS/MS quantification of these DNA lesions.

Additionally, we characterized the threshold energy of cross-link formation based upon the UVB irradiation of ODNs harboring a 5-halouracil or 5-halocytosine (Chapter 3). We found that the calculated threshold energies for 5-halocytosines were more
exothermic than the corresponding 5-halouracils, and more exothermic for 5-iodopyrimidines than for the 5-bromopyrimidine counterparts, suggesting greater crosslink formation for the former pyrimidine derivatives. These calculation results were in
excellent agreement with reported experimentally determined yields of the cross-link products and supported the mechanistic proposal that cross-link products form from UVinduced
electron transfer followed by heterolytic cleavage of the C5-X bond.

In part II, we focused on the characterization of hTDG activity towards oxidized pyrimidine base modifications formed at methyl-CpG site. We used DFT methods to assess the kinetic and thermodynamic parameters for the cleavage of the N-glycosidic
bond of 5-methyl-2’-deoxycytidine (mdC) and its, (1) oxidation products as it pertains to a newly proposed mechanism for active cytosine demethylation (Chapter 4); and (2) oxidation and subsequent deamination products [i.e., the 2’-deoxyuridine (dU)
derivatives] (Chapter 5). Our studies suggest that the inherent chemistry associated with the nucleophilic cleavage of the N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG towards 5-substituted dC and dU derivatives. These findings provided novel chemical insights into the role TDG may play in maintaining the epigenetic integrity of the CpG site.

The constitutional isomers uridine (U) and pseudouridine (Ψ) cannot be distinguished from each other by simple mass measurements of RNA or its fragments because the conversion of U into Ψ is a “mass-silent” post-transcriptional modification. Here we propose a new mass spectrometry based method for identification, localization, and relative quantitation of Ψ in RNA consisting of ~20 nucleotides that does not require chemical labeling. Our approach takes advantage of the different fragmentation behavior of uridine (N-glycosidic bond) and pseudouridine (C-glycosidic bond) residues in RNA upon collisionally activated dissociation.

B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) density functional
theory calculations were employed to explore the kinetics and thermodynamics of gas-phase N-glycosidic bond cleavage induced by nucleophilic attack of C1′ with a hydroxide ion in 5-substituted 2′-deoxycytidines. The results showed that, among the 5-substituted 2′-deoxycytidine derivatives examined [XdC, where X = H (dC), CH3 (medC), CH2OH (hmdC), CHO (fmdC), COOH
(cadC), F (FdC), or Br (BrdC)], fmdC and cadC exhibited the lowest energy barrier and largest exothermicity for N-glycosidic bond cleavage. These results paralleled previously reported nucleobase excision activities of human thymine DNA glycosylase (hTDG) toward duplex DNA substrates harboring a thymine and 5-substituted cytosine derivatives when paired with a guanine. Our study suggests that the inherent chemistry associated with the nucleophilic cleavage of N-glycosidic bond constitutes a major factor contributing to the selectivity of hTDG toward 5-substituted dC derivatives. These findings provided novel insights into the role of TDG in active cytosine demethylation.

The primary structure of 3′-imino[60]fulleryl-3′-deoxythymidine ions is studied using mass spectrometry both in the positive and negative modes. Interaction between the subunits is discussed using collision-induced dissociation (CID) spectra. Collisional activation with argon of the sodiated cations leads to the cleavage of the glycosidic bond and the transfer of a radical hydrogen from the deoxyribose to the thymine. The sodiated thymine is the only fragment observed for low collision energies in the positive mode. In the negative mode, two different ionization mechanisms take place, reduction and deprotonation in the presence of triethylamine. The 2.7 eV electron affinity of C60 and its huge cross section compared to the small cross section and predicted 0.44 eV electron affinity of the thymidine subunit most likely localize the radical electron on the fullerene. On the other hand, deprotonation of the 3′-azido-3′-deoxythymidine (AZT) is known to occur in N-3, the most acidic site of the nucleobase. Consequently, deprotonation causes the negative charge to be initially localized on the thymine. Both types of parent anions give the radical anion C60•− as fragment. The other fragments detected are the dehydrogenated 3′-imino[60]fulleryl-3′-deoxyribose anion, C60NH2−, C60N− and C60H−. Since in negative ion mass spectrometry all fragments include the [60]fullerene unit, this suggests that the fragmentation is driven by the electron affinity of the [60]fullerene, likely responsible for a charge transfer between the deprotonated thymine and the C60. Copyright © 2007 John Wiley & Sons, Ltd.