Scholarly Journals--Published

  • Darwanto A, Farrel A, Rogstad D K, & Sowers L C. (2009). Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analytical Biochemistry, 394(1), 13-23. The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3' and 5' ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple beta-elimination product as described previously. On the basis of (18)O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair. Published by Elsevier Inc. (11/2009) (link)
  • Theruvathu J A, Kim C H, Rogstad D K, Neidigh J W, & Sowers L C. (2009). Base Pairing Configuration and Stability of an Oligonucleotide Duplex Containing a 5-Chlorouracil-Adenine Base Pair. Biochemistry, 48(31), 7539-7546. Inflammation-mediated reactive molecules can damage DNA by oxidation and chlorination. The biological consequences of this damage are as yet incompletely understood. In this paper, we have constructed oligonucleotides containing 5-chlorouracil (ClU), one of the known inflammation damage products. The thermodynamic stability, base pairing configuration, and duplex conformation of oligonucleotides containing ClU paired opposite adenine have been examined. NMR spectra reveal that the ClU-A base pair adopts a geometry similar to that of the T-A base pair, and the ClU-A base pair-containing duplex adopts a normal B-form conformation. The line width of the imino proton of the ClU residue is substantially greater than that of the corresponding T imino proton; however, this difference is not attributed to a reduced thermal or thermodynamic stability or to an increased level of proton exchange with solvent. While the NMR studies reveal an increased level of chemical exchange for the ClU imino proton of the ClU-A base pair, the ClU residue is not a target for removal by the Escherichia coli mispaired uracil glycosylase, which senses damage-related helix instability. The results of this study are consistent with previous reports indicating that the DNA of replicating cells can tolerate substantial substitution with ClU. The fraudulent, pseudo-Watson-Crick ClU-A base pair IS Sufficiently stable to avoid glycosylase removal and, therefore, might constitute a persistent form of cellular DNA damage. (08/2009) (link)
  • Rogstad D K, Herring J L, Theruvathu J A, Burdzy A, Perry C C, Neidigh J W, & Sowers L C. (2009). Chemical Decomposition of 5-Aza-2 '-deoxycytidine (Decitabine): Kinetic Analyses and Identification of Products by NMR, HPLC, and Mass Spectrometry. Chemical Research in Toxicology, 22(6), 1194-1204. The nucleoside analogue 5-aza-2'-deoxycytidine (Decitabine, DAC) is one of several drugs in clinical use that inhibit DNA methyltransferases, leading to a decrease of 5-methylcytosine in newly replicated DNA and subsequent transcriptional activation of genes silenced by cytosine methylation. In addition to methyl transferase inhibition. DAC has demonstrated toxicity and potential mutagenicity, and can induce a DNA-repair response. The mechanisms accounting for these events are not well understood. DAC is chemically unstable in aqueous solutions, but there is little consensus between previous reports as to its half-life and corresponding products of decomposition at physiological temperature and pH, potentially confounding studies on its mechanism of action and long-term use in humans. Here, we have employed a battery of analytical methods to estimate kinetic rates and to characterize DAC decomposition products under conditions of physiological temperature and pH. Our results indicate that DAC decomposes into a plethora of products, formed by hydrolytic opening and deformylation of the triazine ring, in addition to anomerization and possibly other changes in the sugar ring structure. We also discuss the advantages and problems associated with each analytical method used. The results reported here will facilitate ongoing studies and clinical trials aimed at understanding the mechanisms of action, toxicity, and possible mutagenicity of DAC and related analogues. (06/2009) (link)
  • Darwanto A, Theruvathu J A, Sowers J L, Rogstad D K, Pascal T, Goddard W A, & Sowers L C. (2009). Mechanisms of Base Selection by Human Single-stranded Selective Monofunctional Uracil-DNA Glycosylase. Journal of Biological Chemistry, 284(23), 15835-15846. hSMUG1 (human single-stranded selective monofunctional uracil-DNA glyscosylase) is one of three glycosylases encoded within a small region of human chromosome 12. Those three glycosylases, UNG (uracil-DNA glycosylase), TDG (thymine-DNA glyscosylase), and hSMUG1, have in common the capacity to remove uracil from DNA. However, these glycosylases also repair other lesions and have distinct substrate preferences, indicating that they have potentially redundant but not overlapping physiological roles. The mechanisms by which these glycosylases locate and selectively remove target lesions are not well understood. In addition to uracil, hSMUG1 has been shown to remove some oxidized pyrimidines, suggesting a role in the repair of DNA oxidation damage. In this paper, we describe experiments in which a series of oligonucleotides containing purine and pyrimidine analogs have been used to probe mechanisms by which hSMUG1 distinguishes potential substrates. Our results indicate that the preference of hSMUG1 for mispaired uracil over uracil paired with adenine is best explained by the reduced stability of a duplex containing a mispair, consistent with previous reports with Escherichia coli mispaired uracil-DNA glycosylase. We have also extended the substrate range of hSMUG1 to include 5-carboxyuracil, the last in the series of damage products from thymine methyl group oxidation. The properties used by hSMUG1 to select damaged pyrimidines include the size and free energy of solvation of the 5-substituent but not electronic inductive properties. The observed distinct mechanisms of base selection demonstrated for members of the uracil glycosylase family help explain how considerable diversity in chemical lesion repair can be achieved. (06/2009) (link)
  • Herring J L, Rogstad D K, & Sowers L C. (2009). Enzymatic Methylation of DNA in Cultured Human Cells Studied by Stable Isotope Incorporation and Mass Spectrometry. Chemical Research in Toxicology, 22(6), 1060-1068. Enzymatic methylation of cytosine residues in DNA, in conjunction with covalent histone modifications, establishes an epigenetic code essential for the proper control of gene expression in higher organisms. Once established during cellular differentiation, the epigenetic code must be faithfully transmitted to progeny cells. However, epigenetic perturbations can be found in most if not all cancer cells, and the mechanisms leading to these changes are not well understood. In this paper, we describe a series of experiments aimed at understanding the dynamic process of DNA methylation that follows DNA replication. Cells in culture can be propagated in the presence of (15)N-enriched uridine, which labels the pyrimidine precursor pool as well as newly replicated DNA. Simultaneous culture in the presence of (2)H-enriched methionine results in labeling of newly methylated cytosine residues. An ensemble of 5-methylcytosine residues differing in the degree of isotopic enrichment is generated, which can be examined by mass spectrometry. Using this method, we demonstrate that the kinetics of both DNA replication and methylation of newly replicated DNA are indistinguishable. The majority of methylation following DNA replication is shown to occur on the newly synthesized DNA. The method reported here does, however, suggest an unexpected methylation of parental DNA during DNA replication, which might indicate a previously undescribed chromatin remodeling process. The method presented here will be useful in monitoring the dynamic process of DNA methylation and will allow a more detailed understanding of the mechanisms of clinically used methylation inhibitors and environmental toxicants. (06/2009) (link)