The bisulfite reaction with cytosine and genomic DNA structure.

The bisulfite reaction with native DNA has been extensively employed in the detection of non-B DNA structures that can form spontaneously in DNA. These sequences are dynamic in that they can adopt both normal Watson-Crick paired B-DNA or unusual structures like the Triplex, G-Quadruplex, i-motif and Cruciform or Hairpin. Considerable evidence now suggests that these dynamic sequences play roles in both epigenetics and mutagenesis. The bisulfite reaction with native DNA offers a key approach to their detection. In this application whole cells, isolated nuclei or isolated DNA are treated with bisulfite under non-denaturing conditions in order to detect bisulfite accessible regions DNA that are associated with these structures. Here I review the stereochemistry of the bisulfite reaction, the electronic structure of its DNA cytosine substrates and its application in the detection of unusual structures in native DNA.

i-Motif formation and spontaneous deletions in human cells.

Concatemers of d(TCCC) that were first detected through their association with deletions at the RACK7 locus, are widespread throughout the human genome. Circular dichroism spectra show that d(GGGA)n sequences form G-quadruplexes when n > 3, while i-motif structures form at d(TCCC)n sequences at neutral pH when n ≥ 7 in vitro. In the PC3 cell line, deletions are observed only when the d(TCCC)n variant is long enough to form significant levels of unresolved i-motif structure at neutral pH. The presence of an unresolved i-motif at a representative d(TCCC)n element at RACK7 was suggested by experiments showing that that the region containing the d(TCCC)9 element was susceptible to bisulfite attack in native DNA and that d(TCCC)9 oligo formed an i-motif structure at neutral pH. This in turn suggested that that the i-motif present at this site in native DNA must be susceptible to bisulfite mediated deamination even though it is a closed structure. Bisulfite deamination of the i-motif structure in the model oligodeoxynucleotide was confirmed using mass spectrometry analysis. We conclude that while G-quadruplex formation may contribute to spontaneous mutation at these sites, deletions actually require the potential for i-motif to form and remain unresolved at neutral pH.

Substitution of Cytosine with Guanylurea Decreases the Stability of i-Motif DNA.

Both 5-aza-2'-deoxycytidine (decitabine) and its primary breakdown product, 2'-deoxyriboguanylurea (GuaUre-dR), have been shown to act as mutagens and epimutagens that cause replication stress and alter both DNA methylation and gene expression patterns. As cytosine analogues, both are expected to be preferentially incorporated into regions of GC skew where runs of cytosine residues are sequestered on one strand and guanine residues on the other. Given that such regions have been identified as sites with the potential for effects on gene expression and replication stress linked to formation of alternative DNA secondary structures, it is of interest to determine the influence that these base analogues might have on the stability of structures of this kind. Here we report that incorporation of GuaUre-dR into an i-motif-forming sequence decreases both the thermal and pH stability of an i-motif despite the apparent ability of GuaUre-dR to base pair with cytosine.

Two-stage classifiers that minimize PCA3 and the PSA proteolytic activity testing in the prediction of prostate cancer recurrence after radical prostatectomy.

INTRODUCTION: Early biochemical recurrence after prostate cancer surgery is associated with higher risk of aggressive disease and cancer specific death. Many new tests are being developed that will predict the presence of indicators of aggressive disease like early biochemical recurrence. Since recurrence occurs in less than 10% of patients treated for prostate cancer, validation of such tests will require expensive testing on large patient groups. Moreover, clinical application of the validated test requires that each new patient be tested. In this report we introduce a two-stage classifier system that minimizes the number of patients that must be tested in both the validation and clinical application of any new test for recurrence. MATERIALS AND METHODS: Expressed prostatic secretion specimens were prospectively collected from 450 patients prior to robot-assisted radical prostatectomy for prostate cancer. Patients were followed for 2.5 years for evidence of biochemical recurrence. Standard clinical parameters, the levels proteolytic activity of prostate specific antigen (PSA) and the levels of PCA3 RNA, PSA RNA and TMPRSS2:ERG fusion RNA were determined in each prospective patient specimen for subsequent correlation with biochemical recurrence. RESULTS: While levels of PCA3 and PSA proteolytic activity (PPA) in prostatic secretions provided an effective pre-surgical predictor of early biochemical recurrence in prostate cancer, application of the two-stage classifier shows that only 60% of the patients need these tests. CONCLUSION: Two-stage classifiers can provide a parsimonious approach to both the validation and clinical application of biomarker-based tests. Adoption of the two-stage neutral zone classifier can reduce unnecessary testing in prostate cancer treatment.

Expressed prostatic secretion biomarkers improve stratification of NCCN active surveillance candidates: performance of secretion capacity and TMPRSS2:ERG models.

PURPOSE: Active surveillance is a viable patient option for prostate cancer provided that a clinical determination of low risk and presumably organ confined disease can be made. To standardize risk stratification schemes the NCCN (National Comprehensive Cancer Network®) provides guidelines for the active surveillance option. We determined the effectiveness of expressed prostatic secretion biomarkers for detecting occult risk factors in NCCN active surveillance candidates. MATERIALS AND METHODS: Expressed prostatic secretion specimens were obtained before robot-assisted radical prostatectomy. Secretion capacity biomarkers, including total RNA and expressed prostatic secretion specimen volume, were measured by standard techniques. RNA expression biomarkers, including TXNRD1 mRNA, prostate specific antigen mRNA, TMPRSS2:ERG fusion mRNA and PCA3 mRNA, were measured by quantitative reverse-transcription polymerase chain reaction. RESULTS: Of the 528 patients from whom expressed prostatic secretions were collected 216 were eligible for active surveillance under NCCN guidelines. Variable selection on logistic regression identified 2 models, including one featuring types III and VI TMPRSS2:ERG variants, and one featuring 2 secretion capacity biomarkers. Of the 2 high performing models the secretion capacity model was most effective for detecting cases in this group that were up-staged or up-staged plus upgraded. It decreased the risk of up-staging in patients with a negative test almost eightfold and decreased the risk of up-staging plus upgrading about fivefold while doubling the prevalence of up-staging in the positive test group. CONCLUSIONS: Noninvasive expressed prostatic secretion testing may improve patient acceptance of active surveillance by dramatically reducing the presence of occult risk factors among those eligible for active surveillance under NCCN guidelines.

2'-Deoxyriboguanylurea, the primary breakdown product of 5-aza-2'-deoxyribocytidine, is a mutagen, an epimutagen, an inhibitor of DNA methyltransferases and an inducer of 5-azacytidine-type fragile sites.

5-Aza-2'-deoxycytidine (5azaC-dR) has been employed as an inhibitor of DNA methylation, a chemotherapeutic agent, a clastogen, a mutagen, an inducer of fragile sites and a carcinogen. However, its effects are difficult to quantify because it rapidly breaks down in aqueous solution to the stable compound 2'-deoxyriboguanylurea (GuaUre-dR). Here, we used a phosphoramidite that permits the introduction of GuaUre-dR at defined positions in synthetic oligodeoxynucleotides to demonstrate that it is a potent inhibitor of human DNA methyltransferase 1 (hDNMT1) and the bacterial DNA methyltransferase (M.EcoRII) and that it is a mutagen that can form productive base pairs with either Guanine or Cytosine. Pure GuaUre-dR was found to be an effective demethylating agent and was able to induce 5azaC-dR type fragile sites FRA1J and FRA9E in human cells. Moreover, we report that demethylation associated with C:G → G:C transversion and C:G → T:A transition mutations was observed in human cells exposed to pure GuaUre-dR. The data suggest that most of the effects attributed to 5azaC-dR are exhibited by its stable primary breakdown product.

Genomic Frequencies of Dynamic DNA Sequences and Mammalian Lifespan.

BACKGROUND/AIM: Dynamic DNA sequences (i.e. sequences capable of forming hairpins, G-quadruplexes, i-motifs, and triple helices) can cause replication stress and associated mutations. One example of such a sequence occurs in the RACK7 gene in human DNA. Since this sequence forms i-motif structures at neutral pH that cause replication stress and result in spontaneous deletions in prostate cancer cells, our initial aim was to determine its potential utility as a biomarker of prostate cancer. MATERIALS AND METHODS: We cloned and sequenced the region in RACK7 where i-motif deletions often occur in DNA obtained from eight individuals. Expressed prostatic secretions were obtained from three individuals with a positive biopsy for prostate cancer and two with individuals with a negative biopsy for prostate cancer. Peripheral blood specimens were obtained from two control healthy bone marrow donors and a marrow specimen was obtained from a third healthy marrow donor. Follow-up computer searches of the genomes of 74 mammalian species available at the NCBI ftp site or frequencies of 6 dynamic sequences known to produce mutations or replication stress using a program written in Mathematica were subsequently performed. RESULTS: Deletions were found in RACK7 in specimens from both older normal adults, as well as specimens from older patients with cancer, but not in the youngest normal adult. The deletions appeared to show a weak trend to increasing frequency with patient age. This suggested that endogenous mutations associated with dynamic sequences might accumulate during aging and might serve as biomarkers of biological age rather than direct biomarkers of cancer. To test that hypothesis, we asked whether or not the genomic frequencies of several dynamic sequences known to produce replication stress or mutations in human DNA were inversely correlated with maximum lifespan in mammals. CONCLUSION: Our results confirm this correlation for six dynamic sequences in 74 mammalian genomes studied, thereby suggesting that spontaneously induced replication stress and mutations linked to dynamic sequence frequency may limit lifespan by limiting genome stability.

Duplex DNA from Sites of Helicase-Polymerase Uncoupling Links Non-B DNA Structure Formation to Replicative Stress.

BACKGROUND: Replication impediments can produce helicase-polymerase uncoupling allowing lagging strand synthesis to continue for as much as 6 kb from the site of the impediment. MATERIALS AND METHODS: We developed a cloning procedure designed to recover fragments from lagging strand near the helicase halt site. RESULTS: A total of 62% of clones from a p53-deficient tumor cell line (PC3) and 33% of the clones from a primary cell line (HPS-19I) were within 5 kb of a G-quadruplex forming sequence. Analyses of a RACK7 gene sequence, that was cloned multiple times from the PC3 line, revealed multiple deletions in region about 1 kb from the cloned region that was present in a non-B conformation. Sequences from the region formed G-quadruplex and i-motif structures under physiological conditions. CONCLUSION: Defects in components of non-B structure suppression systems (e.g. p53 helicase targeting) promote replication-linked damage selectively targeted to sequences prone to G-quadruplex and i-motif formation.

Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR.

Many methods for the detection of genomic DNA methylation states have appeared. Currently, nearly all such methods employ bisulfite-mediated deamination of denatured DNA. While this treatment effectively deaminates cytosines to uracils, leaving most 5-methylcytosines intact, it also introduces abasic sites that generate a significant number of single-strand breaks in DNA. We have investigated the interplay of these two processes in order to determine their relative effects on the methylation-sensitive QPCR method. The extent of cleavage of the input DNA is significant and appears to be an increasing function of DNA concentration. Even so, the results suggest that only approximately 10% of a 62-nt target will be lost due to degradation and targets up to 131 nt will suffer only a 20% loss. More significant losses were found to occur during the subsequent removal of bisulfite and desulfonation steps that appear to be the result of size selectivity associated with matrix binding and elution required prior to QPCR in the most commonly used protocols. For biospecimens yielding <1 microg of DNA, these findings suggest that bisulfite treatment, in current implementations of MS-QPCR, result in low recoveries that preclude reliable analysis of DNA methylation patterns regardless of target size.

Performance of a single assay for both type III and type VI TMPRSS2:ERG fusions in noninvasive prediction of prostate biopsy outcome.

BACKGROUND: TMPRSS2:ERG fusions are promising prostate cancer biomarkers. Because they can occur in multiple forms in a single cancer specimen, we developed a quantitative PCR test that detects both type III and type VI TMPRSS2:ERG fusions. The assay is quantified from a standard curve determined with a plasmid-cloned type III TMPRSS2:ERG fusion target. METHODS: We collected expressed prostatic secretion (EPS) under an institutional review board-approved, blinded, prospective study from 74 patients undergoing transrectal ultrasound-guided biopsy for prostate cancer. We compared the characteristic performance of the test for type III and type VI TMPRSS2:ERG fusions in predicting biopsy outcome and distinguishing between high and low Gleason scores with similar tests for the expression of PCA3 and DNA methylation levels of the APC, RARB, RASSF1, and GSTP1 genes. We used logistic regression to analyze the effects of multiple biomarkers in linear combinations. RESULTS: Each test provided a significant improvement in characteristic performance over baseline digital rectal examination (DRE) plus serum prostate-specific antigen (PSA); however, the test for type III and type VI TMPRSS2:ERG fusions yielded the best performance in predicting biopsy outcome [area under the curve (AUC) 0.823, 95% CI 0.728-0.919, P < 0.001] and Gleason grade >7 (AUC 0.844, 95% CI 0.740-0.948, P < 0.001). CONCLUSIONS: Although each test appears to have diagnostic value, PSA plus DRE plus type III and type VI TMPRSS2:ERG provided the best diagnostic performance in EPS specimens.

Secondary structure at a hot spot for DNA methylation in DNA from human breast cancers.

The VNTR at c-Ha-ras resides in a hotspot for DNA methylation on chromosome 11 in human tumors, where it is flanked by two MspI restriction sites. We have investigated the nature of the MspI site polymorphism at the c-Ha-ras VNTR observed in variety of tumors including breast cancer.We find that the MspI site 5' to the VNTR is present in a Non-B DNA structure with single-strand character that renders it accessible to bisulfite modification under native conditions, while the MspI site 3' to the VNTR appears to reside in a normal B-form structure that is inaccessible to bisulfite. The non-B DNA structure accounts for the observed polymorphism since MspI cannot cleave single-stranded DNA and control experiments show that the MspI sites were neither mutated nor abnormally methylated. Southern blotting showed that structural polymorphism was present in tumor DNA and tumor adjacent normal tissue DNA but absent from lymphocyte DNA from the same patients. We conclude that the non-B DNA structural polymorphism detected in human tumors near the c-Ha-ras VNTR is a self-perpetuating epigenetic mark that manifests itself spontaneously during breast carcinogenesis in a methylation hot spot.

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells.

Several reports suggest that C(m)CWGG methylation tends not to co-exist with (m)CG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII-GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C(m)CWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C(m)CWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C(m)CWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C(m)CWGG in its promoter. Kinetic studies suggested that an adjacent C(m)CWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C(m)CWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

Gastric DNA damage through tobacco chewing: in vitro mechanistic studies of DNA nitrite attack.

Smokeless chewing tobacco or snuff has been linked to carcinogenic effects in upper aerodigestive organs. The presence of nitrite within the tobacco product is suspected to foster carcinogenic DNA mechanisms at lower pH. We studied the impact of sodium nitrite on DNA damage at single-strand conformers or hairpin loops, known to be present at fragile sites that have been shown to cause methyltransferase stalling and that can lead to chromosomal breakage. At a pH of 4.2, two base-damage products could be demonstrated at significant levels (1-5% of total nucleotides), with greater sensitivity to hairpin loops compared to a control Watson-Crick duplex. Pyrimidine-rich strands (CCG, CTG) were more reactive than purine-rich strands (CAG, CGG). The data support a mechanism for allele-specific predisposition to DNA damage. This mechanism may be of significance in gastric cancer initiation due to chewing tobacco.

Presurgical Biomarker Performance in the Detection of Gleason Upgrading in Prostate Cancer.

BACKGROUND: Gleason Score (GS) upgrading is generally considered a trigger for exit to definitive treatment during active surveillance (AS). Predicting the potential for GS upgrading would be of value in assessing AS eligibility. METHODS: We assessed the performance of biomarkers in presurgical specimens of expressed prostatic secretion (EPS) in this setting. RESULTS: Although EPS volume, total recovered RNA, and RNA expression biomarkers (TMPRSS2: ERG, PCA3, PSA) have been successful in both biopsy outcome prediction, and in the prediction of upstaging in active surveillance eligible patients, they were unable to predict upgrading in patients eligible for active surveillance under National Comprehensive Cancer Network guidelines. CONCLUSIONS: These biomarkers do not improve the prediction of upgrading over indications from standard clinical parameters. IMPACT: Additional biomarkers will be needed in this area. Cancer Epidemiol Biomarkers Prev; 25(12); 1643-5. ©2016 AACR.

New insight into formation of DNA-containing microparticles during PCR: the scaffolding role of magnesium pyrophosphate crystals.

This work aims to study molecular mechanisms involved in the formation of DNA-containing microparticles and nanoparticles during PCR. Both pyrophosphate and Mg(2+) ions proved to play an important role in the generation of DNA microparticles (MPs) with a unique and sophisticated structure in PCR with Taq polymerase. Thus, the addition of Tli thermostable pyrophosphatase to a PCR mixture inhibited this process and caused the destruction of synthesized DNA MPs. Thermal cycling of Na-pyrophosphate (Na-PPi)- and Mg(2+)-containing mixtures (without DNA polymerase and dNTPs) under the standard PCR regime yielded crystalline oval or lenticular microdisks and 3D MPs composed from magnesium pyrophosphate (Mg-PPi). As shown by scanning electron microscopy (SEM), the produced Mg-PPi microparticles consisted of intersecting disks or their segments. They were morphologically similar but simpler than DNA-containing MPs generated in PCR. The incorporation of dNTPs, primers, or dsDNA into Mg-pyrophosphate particles resulted in the structural diversification of 3D microparticles. Thus, the unusual and complex structure of DNA MPs generated in PCR is governed by the unique feature of Mg-pyrophosphate to form supramolecular particles during thermal cycling. We hypothesize the Mg-pyrophosphate particles that are produced during thermal cycling serve as scaffolds for amplicon DNA condensation.

Construction of ordered protein arrays.

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

Design and analysis of nanoscale bioassemblies.

Bionanotechnology is an emerging field in nanotechnology. In general, it uses concepts from chemistry, biochemistry, and molecular biology to identify components and processes for the construction of self-assembling materials and devices. Distant goals of the science of bionanotechnology range from developing programmable nanoscale devices that can sample or alter their environments to developing assemblies capable of Darwinian evolution. At the heart of these approaches is the concept of the production of supramolecular assemblies (SMAs; also known as supramolecular aggregates) by programmed self-assembly in an aqueous medium. Ordered arrays, planar and closed-shell tilings, dynamic machines, and switches have been designed and constructed by using DNA-DNA, protein-protein, and protein-nucleic acid biospecificities. We review the designs and the analytical techniques that have been employed in the production of SMAs that do not occur in nature.

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR.

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg(2+)-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils - that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg(2+) - mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.

Maintaining the unmethylated state.

BACKGROUND: A remarkable correspondence exists between the cytogenetic locations of the known fragile sites and frequently reported sites of hypermethylation. The best-known features of fragile sites are sequence motifs that are prone to the spontaneous formation of a non-B DNA structure. These facts, coupled with the known enzymological specificities of DNA methyltransferase 1 (DNMT1), the ATP-dependent and actin-dependent helicases, and the ten-eleven translocation (TET) dioxygenases, suggest that these enzymes are involved in an epigenetic cycle that maintains the unmethylated state at these sites by resolving non-B structure, preventing both the sequestration of DNA methyltransferases (DNMTs) and hypermethylation in normal cells. PRESENTATION OF THE HYPOTHESIS: The innate tendency of DNA sequences present at fragile sites to form non-B DNA structures results in de novo methylation of DNA at these sites that is held in check in normal cells by the action of ATP-dependent and actin-dependent helicases coupled with the action of TET dioxygenases. This constitutes a previously unrecognized epigenetic repair cycle in which spontaneously forming non-B DNA structures formed at fragile sites are methylated by DNMTs as they are removed by the action of ATP-dependent and actin-dependent helicases, with the resulting nascent methylation rendered non-transmissible by TET dioxygenases. TESTING THE HYPOTHESIS: A strong prediction of the hypothesis is that knockdown of ATP-dependent and actin-dependent helicases will result in enhanced bisulfite sensitivity and hypermethylation at non-B structures in multiple fragile sites coupled with global hypomethylation. IMPLICATIONS OF THE HYPOTHESIS: A key implication of the hypothesis is that helicases, like the lymphoid-specific helicase and alpha thalassemia/mental retardation syndrome X-linked helicase, passively promote accurate maintenance of DNA methylation by preventing the sequestration of DNMTs at sites of unrepaired non-B DNA structure. When helicase action is blocked due to mutation or downregulation of the respective genes, DNMTs stall at unrepaired non-B structures in fragile sites after methylating them and are unable to methylate other sites in the genome, resulting in hypermethylation at non-B DNA-forming sites, along with hypomethylation elsewhere.