Annalisa Masi

Structural basis for the recognition of diastereomeric 5′,8-cyclo-2′-deoxypurine lesions by the human nucleotide excision repair system

The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5′R cyclopurines were greater by a factor of ∼2 than for the 5′S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5′R than in 5′S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5′R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5′,8 bonds.

A 5′, 8-cyclo-2′-deoxypurine lesion induces trinucleotide repeat deletion via a unique lesion bypass by DNA polymerase β

5′,8-cyclo-2′-deoxypurines (cdPus) are common forms of oxidized DNA lesions resulting from endogenous and environmental oxidative stress such as ionizing radiation. The lesions can only be repaired by nucleotide excision repair with a low efficiency. This results in their accumulation in the genome that leads to stalling of the replication DNA polymerases and poor lesion bypass by translesion DNA polymerases. Trinucleotide repeats (TNRs) consist of tandem repeats of Gs and As and therefore are hotspots of cdPus. In this study, we provided the first evidence that both (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) in a CAG repeat tract caused CTG repeat deletion exclusively during DNA lagging strand maturation and base excision repair. We found that a cdA induced the formation of a CAG loop in the template strand, which was skipped over by DNA polymerase β (pol β) lesion bypass synthesis. This subsequently resulted in the formation of a long flap that was efficiently cleaved by flap endonuclease 1, thereby leading to repeat deletion. Our study indicates that accumulation of cdPus in the human genome can lead to TNR instability via a unique lesion bypass by pol β.

Fatty Acids in Membranes as Homeostatic, Metabolic and Nutritional Biomarkers: Recent Advancements in Analytics and Diagnostics

Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress—with an excess of radical and oxidative processes—cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.

Delocalized hole domains in Guanine-rich DNA oligonucleotides.

Differential pulse voltammetries of guanine-rich single- and double-stranded oligonucleotides containing up to six consecutive guanines are reported. The observed progressive lowering of the first voltammetric peak potential as the number of adjacent guanines increases unambiguously points toward the establishment of delocalized hole domains; the hole stabilization energy is ca. 0.1 eV per GG step, significantly lower than that observed for AA steps.

Inhibition of Amyloid Peptide Fragment Aβ25–35 Fibrillogenesis and Toxicity by N-Terminal β-Amino Acid-Containing Esapeptides: Is Taurine Moiety Essential for In Vivo Effects?

We report the synthesis and fibrillogenesis inhibiting activity of the new peptide derivatives 1–4, related to the pentapeptide Ac‐LPFFD‐NH2 (iAβ5p), proposed by Soto and co‐workers and widely recognized as one of the most active β‐sheet breaker agents. The Aβ25–35 fragment of the parent full‐length Aβ1–42 was used as fibrillogenesis model. The activity of peptide derivatives 1–4 was tested in vitro by thioflavin T binding assay, far UV CD spectroscopy, and scanning electron microscopy. Their ability to hinder the toxic effect of Aβ25–35in vivo was studied by monitoring the viability of human SH‐SY5Y neuroblastoma cells and the prevention of superoxide anion radical release from BV2 microglial cells. The results point to a favourable role in the fibrillogenesis inhibitory activity of the sulphonamide junction for compounds 1 and 2, containing an N,N‐dimethyltaurine and a taurine amino‐terminal moiety, respectively. Furthermore, compounds 1 and 2 show a significant protective effect on cell viability, rescuing the cells from the toxicity exerted by Aβ25–35 treatment.

A problem solving approach for the diastereoselective synthesis of (5′ S )- and (5′ R )-5′,8-cyclopurine lesions

A short, efficient and high-yielding approach for the diastereoselective syntheses of the four (5′S)- and (5′R)-5′,8-cyclopurine lesions and their phosphoramidites has been developed. The intermediates prepared by these synthetic pathways provide an easy access to those modified nucleosides that constitute an important molecular library for recognition of DNA damage. With respect to previous synthesis, the key methodologies for cyclization steps and phosphoramidite synthesis were ameliorated.

Biomimetic models of radical stress and related biomarkers.

The biological consequences of free radical production is the central subject of a very lively scientific debate, focusing on the estimation of the type and extent of damage, as well as the efficiency of the protective and repair systems. When studying free radical based chemical mechanisms, it is very important to establish biomimetic models, which allow the experiments to be performed in a simplified environment, but suitably designed to be in strict connection with cellular conditions. The biomimetic modeling approach has been coupled with physical organic chemistry methodologies and knowledge of free radical reactivity. Molecular basis of important processes have been identified, building up molecular libraries of products concerning unsaturated lipids, sulfur-containing proteins and nucleic acids, to be developed as biomarkers. Ongoing projects in our group deal with lipidomics, genomics and proteomics of free radical stress and some examples will be described.

The association constant of 5′,8-cyclo-2′-deoxyguanosine with cytidine

The association of 5′,8-cyclo-2′-deoxyguanosine (cdG), a DNA tandem lesion, with its complementary base cytosine has been studied by voltammetry and NMR in chloroform, using properly silylated derivatives of the two nucleobases for increasing their solubilities. Both voltammetric data and NMR titrations indicated that the Watson-Crick complex of cytidine with cdG is weaker than that with guanosine, the difference being approximately of one order of magnitude between the two association constants.