Biomedical subjects

L C Sowers

Publications and source records attributed to L C Sowers.

At least 19 recordsLinked to original sources

PubMed2004

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83.

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.

DNA Damage↗

PubMed2000

An unexpectedly high excision capacity for mispaired 5-hydroxymethyluracil in human cell extracts.

The oxidation of thymine in DNA can generate a base pair between 5-hydroxymethyluracil (HmU) and adenine, whereas the oxidation and deamination of 5-methylcytosine (5mC) in DNA can generate a base pair between HmU and guanine. Using synthetic oligonucleotides containing HmU at a defined site, HmU-DNA glycosylase activities in HeLa cell and human fibroblast cell extracts have been observed. An HmU-DNA glycosylase activity that removes HmU mispaired with guanine has been measured. Surprisingly, the HmU:G excision activity is 60 times greater than the corresponding HmU:A activity, even though the expected rate of formation of the HmU:A base pair exceeds that of the HmU:G base pair by a factor of 10(7). The HmU:G mispair would arise from the 5mC:G base pair, and, if unrepaired, would give rise to a transition mutation. The observation of an unexpectedly high HmU:G glycosylase activity suggests that human cells may encounter the HmU:G mispair much more frequently than expected. The conversion of 5mC to HmU must be considered as a potential pathway for the generation of 5mC to T transition mutations, which are often found in human tumors.

Adenine↗

PubMed2000

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

Alkaloids↗

PubMed2000

Multiple structures for the 2-aminopurine-cytosine mispair.

The base pair formed between 2-aminopurine (2AP) and cytosine (C) is an intermediate in transition mutations generated by 2AP. To date, several structures have been proposed for the 2AP-C mispair, including those involving a rare tautomer, a protonated base pair, and a neutral wobble structure. In this paper, we describe a series of UV, fluorescence, and NMR studies which demonstrate that an equilibrium exists between the neutral wobble and the protonated Watson-Crick structures. The apparent pK value for the transition between the structures is 5.9-6.0. Formation of a Watson-Crick base pair is accomplished predominantly by protonation of the 2AP residue as indicated by UV spectral changes, fluorescence quenching, and changes in proton chemical shifts. Rapid transfer of the shared proton between the 2AP and cytosine residues is indicated by the rapid exchange of the cytosine amino protons of the protonated Watson-Crick configuration. The relative contribution of the neutral wobble and protonated Watson-Crick configurations to 2AP-induced transition mutations is discussed.

2-Aminopurine↗

PubMed2000

Conformation and proton configuration of pyrimidine deoxynucleoside oxidation damage products in water.

Emerging data strongly suggest that the oxidation of DNA bases can contribute to genomic instability. Structural changes to DNA, induced by base oxidation, may reduce the fidelity of DNA replication and interfere with sequence-specific DNA-protein interactions. We have examined the structures of a series of pyrimidine deoxynucleoside oxidation damage products in aqueous solution. The modified nucleosides studied include the deoxynucleoside derivatives of 5-hydroxyuracil, 5-hydroxycytosine, 5-(hydroxymethyl)uracil, 5-(hydroxymethyl)cytosine, 5-formyluracil, and 5-formylcytosine. The influence of base oxidation on ionization constants, sugar conformation, and tautomeric configuration has been determined on the basis of UV, proton, and nitrogen NMR spectra of the (15)N-enriched derivatives. The potential biological consequences of the structural perturbations resulting from base oxidation are discussed.

DNA Adducts↗

PubMed2000

15N-enriched 5-fluorocytosine as a probe for examining unusual DNA structures.

A new method is presented for the synthesis of oligonucleotides containing 15N-enriched 5-fluorocytosine (FC). Due to the reduced pK of FC, the amino protons of an unpaired FC residue may be observed at lower values of solution pH. The labeled FC residue has been placed as a template base at a model DNA replication fork. The amino protons of the FC residue have been identified in isotope-edited NMR spectra. Data is presented for a template FC residue unpaired, paired with guanine, and mispaired with adenine. These studies demonstrate the utility of labeled FC in examining unusual DNA structures.

DNA↗

PubMed1999

Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2'-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2'-deoxycytidine.

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2'-deoxycytidine (HO5dCyt) in its anionic form. In DNA, this 2'-deoxycytidine analog arises from the oxidation of 2'-deoxycytidine and induces C --> T transitions with 10(2) greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (approximately 1650 cm-1) is enhanced at approximately 65 degrees C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N3-methylcytidine at high pH and N4-methoxy-2'-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO5dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.

DNA↗

PubMed1999

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis.

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.

2-Aminopurine↗

PubMed1999

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.

Animals↗

PubMed1999

Synthesis and utilization of 13C(8)-enriched purines.

A new and more efficient method is presented for the synthesis of 13C(8)-enriched adenine. In addition, we present for the first time the synthesis of 13C(8)-enriched 2-aminopurine and purine. All three analogues have been converted to the corresponding ribonucleosides. The adenine analogue has been further converted to the 2'-deoxy-nucleoside and incorporated into a synthetic oligonucleotide. Data is presented demonstrating the utility of 13C-enrichment in heteronuclear isotope-edited NMR spectra.

Base Sequence↗

PubMed1998

Synthesis and characterization of isotopically enriched pyrimidine deoxynucleoside oxidation damage products.

Oxidative damage to DNA is an established source of genomic instability. In this paper, we describe the synthesis and characterization of several pyrimidine deoxynucleoside oxidation damage products, enriched with stable isotopes. These products include the 2'-deoxynucleoside derivatives of 5-(hydroxymethyl)uracil, 5-formyluracil, 5-hydroxyuracil, 5-(hydroxymethyl)cytosine, 5-formylcytosine, and 5-hydroxycytosine. The common precursor is 2'-deoxy-2"-deutero[1,3-15N]uridine. Additional stable isotopes are added during functional group conversions. Characterization of these derivatives includes mass spectrometry and 1H and 15N NMR spectroscopy. Proton and nitrogen NMR studies reported here allow an examination of the influence of the modification on sugar conformation and tautomeric equilibrium, properties likely to be important in understanding the biological consequences of these DNA damage products.

DNA Adducts↗

PubMed1998

Quantification of 5-(hydroxymethyl)uracil in DNA by gas chromatography/mass spectrometry: problems and solutions.

Oxidation of the thymine methyl group results in the formation of 5-(hydroxymethyl)uracil (HmU). HmU is a recognized endogenous DNA damage product, and HmU levels in DNA are increased by oxidant stress. Previous studies have reported substantially conflicting values for HmU levels in DNA. In studies utilizing postlabeling methods, HmU levels have been reported to be as high as or higher than the levels of some of the more commonly described DNA oxidation damage products such as 8-oxoguanine. In some studies utilizing GC/MS methods, however, HmU has been undetectable. In acid solution, the hydroxymethyl group of HmU can undergo condensation reactions with carboxylic acids, alcohols, and amines. While HmU can be accurately measured by GC/MS, the first step in the preparation of samples for GC/MS analysis is acid hydrolysis of the DNA. Such hydrolysis would be expected to result in substantial derivatization of HmU. We have utilized chemically synthesized oligonucleotides containing a known amount of HmU as well as an isotopically enriched standard to investigate the chemical modification of HmU during the acid hydrolysis of DNA. We conclude that HmU levels reported by GC/MS following acid hydrolysis may be up to an order of magnitude lower than the actual levels. Further, we propose modifications to the standard hydrolysis protocols which maximize recovery of HmU prior to silylation and analysis by GC/MS.

DNA↗

PubMed1997

Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.

Cooperative binding of the bacteriophage lambda cI repressor dimer to specific sites of the phage operators OR and OL controls the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993) Biochemistry 32, 6179-6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor-OR interactions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer-octamer association reactions of a repressor dimer-OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein-DNA interactions.

Bacteriophage lambda↗

PubMed1997

Spectroscopic determination of open complex formation at promoters for Escherichia coli RNA polymerase.

A considerable amount of effort has been expended studying the kinetics of association of Escherichia coli RNA polymerase with promoter DNA in forming the open complex. Strand separation occurs over about 12 base pairs and includes the transcription start site. However, these efforts have been significantly hampered by the lack of a sensitive, real time method by which formation of an open complex could be assayed. Here, we employ short (86 bp) synthetic promoters with 2-aminopurine (2-AP) substitutions in the region that becomes single-stranded to spectroscopically monitor open complex formation. We demonstrate that promoters bearing the substitutions behave in a manner similar to that of those containing only the four common bases with respect to both the region of strand separation and start site selection. Open complex formation was found to yield an increased fluorescence signal with an emission maximum characteristic of 2-aminopurine. This spectroscopic assay for open complex formation was found to be well-suited to the investigation of a strong promoter, allowing open complex formation to be followed over a time scale of seconds with a stopped flow apparatus. The introduction of two additional nonconsensus base pairs in the -35 region resulted in a promoter for which open complex formation was 100-fold slower. The same substrates were also used to monitor the promoter re-annealing that ensues upon initiation of RNA synthesis. Similar rates for this process were observed for the two promoter variants employed in this study.

2-Aminopurine↗

PubMed1997

Solid phase synthesis and restriction endonuclease cleavage of oligodeoxynucleotides containing 5-(hydroxymethyl)-cytosine.

Emerging data suggest an important role for cytosine methylation in tumorigenesis. Simultaneously, recent studies indicate a significant contribution of endogenous oxidative DNA damage to the development of human disease. Oxidation of the 5-methyl group of 5-methylcytosine (5mC) residues in DNA results in the formation of 5-(hydroxymethyl)cytosine (hmC). The biological consequences ofhmC residues in vertebrate DNA are as yet unknown; however, conversion of the hydrophobic methyl group to the hydrophilic hydroxymethyl group may substantially alter the interaction of sequence-specific binding proteins with DNA. Central to both biophysical and biochemical studies on the potential consequences of specific DNA damage products such as hmC are efficient methods for the synthesis of oligodeoxynucleotides containing such modified bases at selected positions. In this paper, we describe a method for the placement of hmC residues in oligodeoxynucleotides using established phosphoramidite chemistry. In addition, we have examined the influence of specific hmC residues on enzymatic cleavage of oligodeoxynucleotides by the methylation-sensitive restriction endonucleases MspI and HpaII.

5-Methylcytosine↗

PubMed1997

Synthesis and cleavage of oligodeoxynucleotides containing a 5-hydroxyuracil residue at a defined site.

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.

Antineoplastic Agents↗

PubMed1996

Methoxyamine-induced mutagenesis of nucleic acids. A proton NMR study of oligonucleotides containing N4-methoxycytosine paired with adenine or guanine.

We report the solution structure of two heptanucleotides each containing a central N4-methoxycytosine, in one case paired with adenine on the opposite strand and the other with guanine. For the N4-methoxycytosine adenine pair, only the imino form of the N4-methoxycytosine residue is observed and base pairing is in Watson-Crick geometry. However, rotation of the methoxy group about the N-OCH3 bond is not constrained to a particular orientation although it must be anti to the N3 of N4-methoxycytosine. The slow exchange on a proton NMR time scale between the single strand and double strand forms is attributed to the strong preference of the cis conformation of the OCH3 group in the single strand, which inhibits base pair formation. For the N4-methoxycytosine that is base paired with guanine, we observe an amino form in Watson-Crick geometry in slow exchange with a base paired imino form in wobble geometry. The amino form is predominant at low temperature whereas the imino form predominates above 313 K. We have measured the exchange rate between the two forms at 303 K and observed a value of approximately 1 S-1. The relative ratio of amino and imino forms of N4-methoxycytosine is influenced by both the base that is in front and the temperature. Our results explain the preferential replacement of dTTP by N4-methoxycytosine in primer elongation.

Adenine↗

PubMed1996

Solution structure of two mismatches G.G and I.I in the K-ras gene context by nuclear magnetic resonance and molecular dynamics.

Two mismatches, G.G and I.I, have been incorporated at the central position of 5'd-(GCCACXAGCTC).d(GAGCTXGTGGC) in order to carry out NMR and molecular dynamics studies. These duplexes constitute the sequence 29-39 of the K-ras gene coding for the glycine 12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For the duplex containing the G.G mismatch, we propose for the major species, a type of pairing involving one hydrogen bond between the imino group of one central guanine and the carbonyl group of the opposite guanine. Both bases are in an anti conformation. Two conformations, with the same donor and acceptor pattern can coexist, one is obtained from the other by a 180 degrees rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature. A minor species involving hydrogen bonding between the guanine amino group and the carbonyl group of the guanine on the opposite strand may also exist as shown by the molecular dynamics calculations. For the I.I mismatch we observe the same major species, i.e., hydrogen bonding between an imino proton of one base and the carbonyl group of the base on the opposite strand with both bases in an anti conformation. Exchange between these two conformations is faster than for the G.G mismatch. Further, we observe that the I.I mismatch adopts a minor conformation, in which one or other of the bases is in the syn conformation.

Chromosome Mapping↗