Transition Mutations in the hTERT Promoter Are Unrelated to Potential i-motif Formation in the C-Rich Strand.

Increased expression of the human telomere reverse transcriptase (hTERT) in tumors promotes tumor cell survival and diminishes the survival of patients. Cytosine-to-thymine (C-to-T) transition mutations (C250T or C228T) in the hTERT promoter create binding sites for transcription factors, which enhance transcription. The G-rich strand of the hTERT promoter can form G-quadruplex structures, whereas the C-rich strand can form an i-motif in which multiple cytosine residues are protonated. We considered the possibility that i-motif formation might promote cytosine deamination to uracil and C-to-T mutations. We computationally probed the accessibility of cytosine residues in an i-motif to attack by water. We experimentally examined regions of the C-rich strand to form i-motifs using pH-dependent UV and CD spectra. We then incubated the C-rich strand with and without the G-rich complementary strand DNA under various conditions, followed by deep sequencing. Surprisingly, deamination rates did not vary substantially across the 46 cytosines examined, and the two mutation hotspots were not deamination hotspots. The appearance of mutational hotspots in tumors is more likely the result of the selection of sequences with increased promoter binding affinity and hTERT expression.

DNA Base Excision Repair Intermediates Influence Duplex-Quadruplex Equilibrium.

Although genomic DNA is predominantly duplex under physiological conditions, particular sequence motifs can favor the formation of alternative secondary structures, including the G-quadruplex. These structures can exist within gene promoters, telomeric DNA, and regions of the genome frequently found altered in human cancers. DNA is also subject to hydrolytic and oxidative damage, and its local structure can influence the type of damage and its magnitude. Although the repair of endogenous DNA damage by the base excision repair (BER) pathway has been extensively studied in duplex DNA, substantially less is known about repair in non-duplex DNA structures. Therefore, we wanted to better understand the effect of DNA damage and repair on quadruplex structure. We first examined the effect of placing pyrimidine damage products uracil, 5-hydroxymethyluracil, the chemotherapy agent 5-fluorouracil, and an abasic site into the loop region of a 22-base telomeric repeat sequence known to form a G-quadruplex. Quadruplex formation was unaffected by these analogs. However, the activity of the BER enzymes were negatively impacted. Uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1) were inhibited, and apurinic/apyrimidinic endonuclease 1 (APE1) activity was completely blocked. Interestingly, when we performed studies placing DNA repair intermediates into the strand opposite the quadruplex, we found that they destabilized the duplex and promoted quadruplex formation. We propose that while duplex is the preferred configuration, there is kinetic conversion between duplex and quadruplex. This is supported by our studies using a quadruplex stabilizing molecule, pyridostatin, that is able to promote quadruplex formation starting from duplex DNA. Our results suggest how DNA damage and repair intermediates can alter duplex-quadruplex equilibrium.

Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of ß-mercaptoethanol (ß-ME), the abasic site unsaturated aldehyde forms a ß-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.

Chemical and enzymatic modifications of 5-methylcytosine at the intersection of DNA damage, repair, and epigenetic reprogramming.

The DNA of all living organisms is persistently damaged by endogenous reactions including deamination and oxidation. Such damage, if not repaired correctly, can result in mutations that drive tumor development. In addition to chemical damage, recent studies have established that DNA bases can be enzymatically modified, generating many of the same modified bases. Irrespective of the mechanism of formation, modified bases can alter DNA-protein interactions and therefore modulate epigenetic control of gene transcription. The simultaneous presence of both chemically and enzymatically modified bases in DNA suggests a potential intersection, or collision, between DNA repair and epigenetic reprogramming. In this paper, we have prepared defined sequence oligonucleotides containing the complete set of oxidized and deaminated bases that could arise from 5-methylcytosine. We have probed these substrates with human glycosylases implicated in DNA repair and epigenetic reprogramming. New observations reported here include: SMUG1 excises 5-carboxyuracil (5caU) when paired with A or G. Both TDG and MBD4 cleave 5-formyluracil and 5caU when mispaired with G. Further, TDG not only removes 5-formylcytosine and 5-carboxycytosine when paired with G, but also when mispaired with A. Surprisingly, 5caU is one of the best substrates for human TDG, SMUG1 and MBD4, and a much better substrate than T. The data presented here introduces some unexpected findings that pose new questions on the interactions between endogenous DNA damage, repair, and epigenetic reprogramming pathways.

A combinatorial system to examine the enzymatic repair of multiply damaged DNA substrates.

DNA damage drives genetic mutations that underlie the development of cancer in humans. Multiple pathways have been described in mammalian cells which can repair this damage. However, most work to date has focused upon single lesions in DNA. We present here a combinatorial system which allows assembly of duplexes containing single or multiple types of damage by ligating together six oligonucleotides containing damaged or modified bases. The combinatorial system has dual fluorescent labels allowing examination of both strands simultaneously, in order to study interactions or competition between different DNA repair pathways. Using this system, we demonstrate how repair of oxidative damage in one DNA strand can convert a mispaired T:G deamination intermediate into a T:A mutation. We also demonstrate that slow repair of a T:G mispair, relative to a U:G mispair, by the human methyl-binding domain 4 DNA glycosylase provides a competitive advantage to competing repair pathways, and could explain why CpG dinucleotides are hotspots for C to T mutations in human tumors. Data is also presented that suggests repair of closely spaced lesions in opposing strands can be repaired by a combination of short and long-patch base excision repair and simultaneous repair of multiply damage sites can potentially lead to lethal double strand breaks.

Sewage Promotes Vibrio vulnificus Growth and Alters Gene Transcription in Vibrio vulnificus CMCP6.

Vibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections. IMPORTANCE Vibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.

An assessment of three methods for extracting bacterial DNA from beach sand.

AIMS: Beach water quality is regulated by faecal indicator bacteria levels, sand is not, despite known human health risk from exposure to beach sand. We compared the performance of three methods to extract bacterial DNA from beach sand as a step toward a standard method. METHODS AND RESULTS: The analytical sensitivity of quantitative polymerase chain reaction (qPCR) for Enterococcus was compared for the slurry (suspension, agitation, membrane filtration of supernatant), versus direct extraction using PowerSoil™ or PowerMax Soil™ kits. The slurry method had the lowest limit of detection at 20-80 gene copies g-1 , recovered significantly more DNA, and the only method that detected Enterococcus by qPCR in all samples; therefore, the only method used in subsequent experiments. The slurry method reflected the spatial variability of Enterococcus in individual transect samples. Mean recovery efficiency of the microbial source tracking marker HF183 from wastewater spiked marine and freshwater beach sand was 100.8% and 64.1%, respectively, but varied, indicating that the mixing protocol needs improvement. CONCLUSIONS: Among the three methods, the slurry method had the best analytical sensitivity and produced extracts that were useful for culture or molecular analysis. SIGNIFICANCE AND IMPACT OF STUDY: Standardization of methods for extraction of bacterial DNA from sand facilitates comparisons among studies, and ultimately contributes to the safety of recreational beaches.

Comparison of DNA Methylation in Vibrio vulnificus Cells Grown in Human Serum with Those Grown in Seawater.

The chromosomal methylation statuses of the highly virulent Vibrio vulnificus strain CMCP6 grown in human serum and in seawater are compared here. Growth in seawater resulted in ∼4 times as much methylation as that in human serum, primarily N4-methylcytosines.

Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments.

Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.

Data disclosure for chemical evaluations.

BACKGROUND: Public disclosure of scientific data used by the government to make regulatory decisions for chemicals is a practical step that can enhance public confidence in the scientific basis of such decisions. OBJECTIVES: We reviewed the U.S. Environmental Protection Agency's (EPA) current practices regarding disclosure of data underlying regulatory and policy decisions involving chemicals, including pesticides. We sought to identify additional opportunities for the U.S. EPA to disclose data and, more generally, to promote broad access to data it uses, regardless of origin. DISCUSSION: We recommend that when the U.S. EPA proposes a regulatory determination or other policy decision that relies on scientific research, it should provide sufficient underlying raw data and information about methods to enable reanalysis and attempts to independently reproduce the work, including the sensitivity of results to alternative analyses. This recommendation applies regardless of who conducted the work. If the U.S. EPA is unable to provide such transparency, it should state whether it had full access to all underlying data and methods. A timely version of submitted data cleared of information about confidential business matters and personal privacy should fully meet the standards of transparency described below, including public access sufficient for others to undertake an independent reanalysis. CONCLUSION: Reliable chemical evaluation is essential for protecting public health and the environment and for ensuring availability of useful chemicals under appropriate conditions. Permitting qualified researchers to endeavor to independently reproduce the analyses used in regulatory determinations of pesticides and other chemicals would increase confidence in the scientific basis of such determinations.

Enhancing credibility of chemical safety studies: emerging consensus on key assessment criteria.

OBJECTIVES: We examined the extent to which consensus exists on the criteria that should be used for assessing the credibility of a scientific work, regardless of its funding source, and explored how these criteria might be implemented. DATA SOURCES: Three publications, all presented at a session of the 2009 annual meeting of the Society for Risk Analysis, have proposed a range of criteria for evaluating the credibility of scientific studies. At least two other similar sets of criteria have recently been proposed elsewhere. DATA EXTRACTION/SYNTHESIS: In this article we review these criteria, highlight the commonalities among them, and integrate them into a list of 10 criteria. We also discuss issues inherent in any attempt to implement the criteria systematically. CONCLUSIONS: Recommendations by many scientists and policy experts converge on a finite list of criteria for assessing the credibility of a scientific study without regard to funding source. These criteria should be formalized through a consensus process or a governmental initiative that includes discussion and pilot application of a system for reproducibly implementing them. Formal establishment of such a system should enable the debate regarding chemical studies to move beyond funding issues and focus on scientific merit.

Scientific and legal perspectives on science generated for regulatory activities.

This article originated from a conference that asked "Should scientific work conducted for purposes of advocacy before regulatory agencies or courts be judged by the same standards as science conducted for other purposes?" In the article, which focuses on the regulatory advocacy context, we argue that it can be and should be. First, we describe a set of standards and practices currently being used to judge the quality of scientific research and testing and explain how these standards and practices assist in judging the quality of research and testing regardless of why the work was conducted. These standards and practices include the federal Information Quality Act, federal Good Laboratory Practice standards, peer review, disclosure of funding sources, and transparency in research policies. The more that scientific information meets these standards and practices, the more likely it is to be of high quality, reliable, reproducible, and credible. We then explore legal issues that may be implicated in any effort to create special rules for science conducted specifically for a regulatory proceeding. Federal administrative law does not provide a basis for treating information in a given proceeding differently depending on its source or the reason for which it was generated. To the contrary, this law positively assures that interested persons have the right to offer their technical expertise toward the solution of regulatory problems. Any proposal to subject scientific information generated for the purpose of a regulatory proceeding to more demanding standards than other scientific information considered in that proceeding would clash with this law and would face significant administrative complexities. In a closely related example, the U.S. Environmental Protection Agency considered but abandoned a program to implement standards aimed at "external" information.

Assessing the reliability and credibility of industry science and scientists.

The chemical industry extensively researches and tests its products to implement product stewardship commitments and to ensure compliance with governmental requirements. In this commentary we argue that a wide variety of mechanisms enable policymakers and the public to assure themselves that studies performed or funded by industry are identified as such, meet high scientific standards, and are not suppressed when their findings are adverse to industry's interests. The more a given study follows these practices and standards, the more confidence one can place in it. No federal laws, rules, or policies express a presumption that scientific work should be ignored or given lesser weight because of the source of its funding. To the contrary, Congress has consistently mandated that agencies allow interested or affected parties to provide information to them and fairly consider that information. All participants in scientific review panels should disclose sources of potential biases and conflicts of interest. The former should be considered in seeking a balanced panel rather than being used as a basis for disqualification. Conflicts of interest generally do require disqualification, except where outweighed by the need for a person's services. Within these constraints, chemical industry scientists can serve important and legitimate functions on scientific advisory panels and should not be unjustifiably prevented from contributing to their work.