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.

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.

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.

Distribution of CWG and CCWGG in the human genome.

Expression of the bacterial CG methyltransferase M*HhaI in mammalian cells appears to generate significant biological effects, while biological effects of the expression of the non-CG methyltransferase M*EcoRII in human cells have not been detected. The association of cytosine methylation with the CG site in mammals is also associated with clustering of CG sites near 5' control regions (CG-islands) of human genes.Moreover spontaneous deamination of 5-methylcytosine at these sites is thought to lead to the well known deficiency of CG sites in genomes where endogenous CG methyltransferases are expressed. Since these associations are generally taken to imply a biological function for the CG dinucleotide that is associated with its selective methylation by endogenous DNA methylation systems, we have asked whether or not CWG or CCWGG sites are clustered in regions flanking human genes and whether or not an overall deficiency of CWG or CCWGG occurs in the human genome. Using build 36.1, of the human genome, we inspected the regions flanking the 28,501 well known gene loci in the human genome. Our analysis confirmed the expected clustering of CG sites near the 5' region of known genes and open reading frames. In contrast to the CG site, neither the CWG site nor the CCWGG site recognized by the bacterial methyltransferase M*EcoRII were clustered in any particular region near known genes and open reading frames. Moreover, neither the CCWGG nor the CWG site was depleted in the human genome, again in sharp contrast to the known genomic deficiency of CpG sites. Our findings suggest that in contrast to CG site recognition, human cytosine methyltransferases recognize CWG and CCWGG only at very low frequency if at all.

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.

Application of Nanoscale Bioassemblies to Clinical Laboratory Diagnostics.

This chapter summarizes progress in several approaches and devices that will improve and augment existing diagnostic techniques. The term bionanotechnology has been used to describe the science that supports the construction of nanoscale bioassemblies. In each of the present applications to diagnostics, bionanotechnological devices play a largely passive role. Cell surface targeting with an antibody, a growth factor, or a small molecule ligand achieves a new level of sophistication, however, it is still a passive approach. While the induced conformational changes associated with the binding of dendrimers or molecular beacons are somewhat more complex responses to the local environment, they are still largely passive mechanistically. Dynamic devices that change color with time of incubation based on the presence or absence of secondary or tertiary cellular markers within a population exhibiting a primary marker would be of considerable utility. Dynamic nanoscale devices of this type await the application of the rules of assembly associated with the scaffolds described earlier and perhaps the discovery and application of new rules of assembly and new scaffolds.

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.

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.

Ultrasonic analysis of peptide- and antibody-targeted microbubble contrast agents for molecular imaging of alphavbeta3-expressing cells.

The goal of targeted ultrasound contrast agents is to significantly and selectively enhance the detection of a targeted vascular site. In this manuscript, three distinct contrast agents targeted to the alphavbeta3 integrin are examined. The alphavbeta3 integrin has been shown to be highly expressed on metastatic tumors and endothelial cells during neovascularization, and its expression has been shown to correlate with tumor grade. Specific adhesion of these contrast agents to alphavbeta3-expressing cell monolayers is demonstrated in vitro, and compared with that of nontargeted agents. Acoustic studies illustrate a backscatter amplitude increase from monolayers exposed to the targeted contrast agents of up to 13-fold (22 dB) relative to enhancement due to control bubbles. A linear dependence between the echo amplitude and bubble concentration was observed for bound agents. The decorrelation of the echo from adherent targeted agents is observed over successive pulses as a function of acoustic pressure and bubble density. Frequency-domain analysis demonstrates that adherent targeted bubbles exhibit high-amplitude narrowband echo components, in contrast to the primarily wideband response from free microbubbles. Results suggest that adherent targeted contrast agents are differentiable from free-floating microbubbles, that targeted contrast agents provide higher sensitivity in the detection of angiogenesis, and that conventional ultrasound imaging techniques such as signal subtraction or decorrelation detection can be used to detect integrin-expressing vasculature with sufficient signal-to-noise.

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.

Methods for the design and analysis of oligodeoxynucleotide-based DNA (cytosine-5) methyltransferase inhibitors.

Several second-generation inhibitors of DNA (cytosine-5) methyltransferases based on studies of modified synthetic oligodeoxynucleoides have been described. As an aid to studies of these inhibitors, we present an electronic structure-based algorithm that can be used as a method for predicting the nature of the expected inhibition by any noncytosine nucleotide target. Targeting by the major human enzyme (hDnmt1) is governed by the presence of a three-nucleotide motif. In hemimethylated DNA, this motif consists of a 5-methylcytosine targeting signal that causes the enzyme to probe the opposite strand for a normally paired guanosine or inosine residue and attempt to methylate the residue 5' to that site. As a demonstration of the method, we apply these rules to the design and characterization of a novel oligodeoxynucleotide inhibitor of hDnmt1. This inhibitor takes advantage of the three-nucleotide recognition motif characteristic of hDnmt1 and shows that the enzyme is inhibited in vitro by non-CG methylation which targets the enzyme to normally basepaired but unproductive nucleotides such as dG, dA, and dT. Kinetic analysis at constant S-adenosyl-L-methionine concentration shows that representative inhibitory oligodeoxynucleotides are best viewed as weakly productive components of systems containing two DNA substrates. This model suggests that the most effective inhibitors are those with very low apparent Vmax and very low Km values. Oligodeoxynucleotides containing mispaired and unproductive targets such as dG, dA, dT, and dU are also inhibitory as secondary substrates for the human enzyme. Biologically, fail-safe mechanisms identified by the ab initio approach appear to be active in preventing potentially mutagenic deamination of dihydrocytosine and enzymatic methylation of dU.

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.