M-DNA is a complex between the divalent metal ions Zn2+, Ni2+ and Co2+ and duplex DNA which forms at a pH of approximately 8.5. The stability and formation of M-DNA was monitored with an ethidium fluorescence assay in order to assess the relationship between pH, metal ion concentration, DNA concentration and the base composition. The dismutation of calf thymus DNA exhibits hysteresis with the formation of M-DNA occurring at a higher pH than the reconversion of M-DNA back to B-DNA. Hysteresis is most prominent with the Ni form of M-DNA where complete reconversion to B-DNA takes several hours even in the presence of EDTA. Increasing the DNA concentration leads to an increase in the metal ion concentration required for M-DNA formation. Both poly(dG)*poly(dC) and poly(dA)*poly(dT) formed M-DNA more readily than the corresponding mixed sequence DNAs. For poly(dG)*(poly(dC) M-DNA formation was observed at pH 7.4 with 0.5 mM ZnCl2. Modified bases were incorporated into a 500 bp fragment of phage lambda DNA by polymerase chain reaction. DNAs in which guanine was replaced with hypoxanthine or thymine with 5-fluorouracil formed M-DNA at pHs below 8 whereas substitutions such as 2-aminoadenine and 5-methylcytosine had little effect. Poly[d(A5FU)] also formed a very stable M-DNA duplex as judged from T(m) measurements. It is evident that the lower the pK(a) of the imino proton of the base, the lower the pH at which M-DNA will form; a finding that is consistent with the replacement of the imino proton with the metal ion.

The eMERGE Consortium* , * The advancement of precision medicine requires new methods to coordinate and deliver genetic data from heterogeneous sources to physicians and patients. The eMERGE III Network enrolled >25,000 participants from biobank and prospective cohorts of predominantly healthy individuals for clinical genetic testing to determine clinically actionable findings. The network developed protocols linking together the 11 participant collection sites and 2 clinical genetic testing laboratories. DNA capture panels targeting 109 genes were used for testing of DNA and sample collection, data generation, interpretation, reporting, delivery, and storage were each harmonized. A compliant and secure network enabled ongoing review and reconciliation of clinical interpretations, while maintaining communication and data sharing between clinicians and investigators. A total of 202 individuals had positive diagnostic findings relevant to the indication for testing and 1,294 had additional/secondary findings of medical significance deemed to be returnable, establishing data return rates for other testing endeavors. This study accomplished integration of structured genomic results into multiple electronic health record (EHR) systems, setting the stage for clinical decision support to enable genomic medicine. Further, the established processes enable different sequencing sites to harmonize technical and interpretive aspects of sequencing tests, a critical achievement toward global standardization of genomic testing. The eMERGE protocols and tools are available for widespread dissemination.

Over time and under stressing conditions proteins are susceptible to a variety of spontaneous covalent modifications. One of the more commonly occurring types of protein damage is deamidation; the conversion of asparagines into aspartyls and isoaspartyls. The physiological significance of isoaspartyl formation is emphasized by the presence of the conserved enzyme L-isoaspartyl O-methyltransferase (PIMT), whose physiological function appears to be in preventing the accumulation of deamidated proteins. Seemingly consistent with a repair function, overexpression of PIMT in Drosophila melanogaster extends lifespan under conditions expected to contribute to protein damage. Based on structural information and sequence homology we have created mutants of residues proposed to be involved in co-factor binding in Escherichia coli PIMT. Both mutants retain S-adenosyl L-methionine binding capabilities but demonstrate dramatically reduced kinetic capabilities, perhaps suggestive of catalytic roles beyond co-factor binding. As anticipated, overexpression of the wild type enzyme in E. coli results in bacteria with increased tolerance to thermal stress. Surprisingly, even greater levels of heat tolerance were observed with overexpression of the inactive PIMT mutants. The increased survival capabilities observed with overexpression of PIMT in E. coli, and possibly in Drosophila, are not due to increased isoaspartyl repair capabilities but rather a temperature-independent induction of the heat shock system as a result of overexpression of a misfolding-prone protein.An alternate hypothesis as to the physiological substrate and function of L-isoaspartyl methyltransferase is proposed.Proteins are susceptible to a variety of spontaneous, covalent modifications that have the potential for disruption of both structure and biological activity. The formation of isoaspartyl residues, through either the deamidation of asparagines or isomerization of aspartates, are among the most rapidly occurring types of damage that afflict proteins under physiological conditions (1).The metamorphosis of asparagine and aspartate residues is initiated by the nucleophilic attack of the neighboring peptide nitrogen on the side chain carbonyl, resulting in the cyclization of the side chain with the main chain to the formation of a succinimide ring (Fig.

A 50-base Guide strand was synthesized that consisted of a central 10-base probe sequence flanked by two tracts of 20 adenine residues. Target sequences of 10 bases containing up to three mismatches were prepared and hybridized to the Guide strand in 1 M KCl. The transport of these constructs through single alpha-hemolysin pores was analysed by measuring the current blockade as a function of time. Complementary dsDNA takes significantly longer (840 +/- 60 micro s) to pass through the pore than a sequence of the same length containing a single (590 +/- 45 micro s) and a double (270 +/- 50 micro s) mismatch. Constructs involving three mismatches were indistinguishable from Guide ssDNA transport (120 +/- 30 micro s). The results suggest that dsDNA must unzip as it is transported through the nanopore. Duplexes containing mismatches unzip more quickly and can be distinguished from those with perfect complementarity.

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