FANCD2 and RAD51 recombinase directly inhibit DNA2 nuclease at stalled replication forks and FANCD2 acts as a novel RAD51 mediator in strand exchange to promote genome stability.

FANCD2 protein, a key coordinator and effector of the interstrand crosslink repair pathway, is also required to prevent excessive nascent strand degradation at hydroxyurea-induced stalled forks. The RAD51 recombinase has also been implicated in regulation of resection at stalled replication forks. The mechanistic contributions of these proteins to fork protection are not well understood. Here, we used purified FANCD2 and RAD51 to study how each protein regulates DNA resection at stalled forks. We characterized three mechanisms of FANCD2-mediated fork protection: (1) The N-terminal domain of FANCD2 inhibits the essential DNA2 nuclease activity by directly binding to DNA2 accounting for over-resection in FANCD2 defective cells. (2) Independent of dimerization with FANCI, FANCD2 itself stabilizes RAD51 filaments to inhibit multiple nucleases, including DNA2, MRE11 and EXO1. (3) Unexpectedly, we uncovered a new FANCD2 function: by stabilizing RAD51 filaments, FANCD2 acts to stimulate the strand exchange activity of RAD51. Our work biochemically explains non-canonical mechanisms by which FANCD2 and RAD51 protect stalled forks. We propose a model in which the strand exchange activity of FANCD2 provides a simple molecular explanation for genetic interactions between FANCD2 and BRCA2 in the FA/BRCA fork protection pathway.

Enzymatic bypass of an N6-deoxyadenosine DNA-ethylene dibromide-peptide cross-link by translesion DNA polymerases.

Unrepaired DNA-protein cross-links, due to their bulky nature, can stall replication forks and result in genome instability. Large DNA-protein cross-links can be cleaved into DNA-peptide cross-links, but the extent to which these smaller fragments disrupt normal replication is not clear. Ethylene dibromide (1,2-dibromoethane) is a known carcinogen that can cross-link the repair protein O6-alkylguanine-DNA alkyltransferase (AGT) to the N6 position of deoxyadenosine (dA) in DNA, as well as four other positions in DNA. We investigated the effect of a 15-mer peptide from the active site of AGT, cross-linked to the N6 position of dA, on DNA replication by human translesion synthesis DNA polymerases (Pols) η, ⍳, and κ. The peptide-DNA cross-link was bypassed by the three polymerases at different rates. In steady-state kinetics, the specificity constant (kcat/Km) for incorporation of the correct nucleotide opposite to the adduct decreased by 220-fold with Pol κ, tenfold with pol η, and not at all with Pol ⍳. Pol η incorporated all four nucleotides across from the lesion, with the preference dT > dC > dA > dG, while Pol ⍳ and κ only incorporated the correct nucleotide. However, LC-MS/MS analysis of the primer-template extension product revealed error-free bypass of the cross-linked 15-mer peptide by Pol η. We conclude that a bulky 15-mer peptide cross-linked to the N6 position of dA can retard polymerization and cause miscoding but that overall fidelity is not compromised because only correct pairs are extended.

Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases.

1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2'-deoxyadenosine and 2'-deoxyguanosine ( Cmarik , J. L. , et al. ( 1991 ) J. Biol. Chem. 267 , 6672 - 6679 ). Adduction at the N6 position of 2'-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O6-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. Chem. Int. Ed. 52 , 12879 - 12882 ). We identified and quantified a new adduct, S-[2-(N6-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols η and ι, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol η and ι showed increased misincorporation opposite the adduct compared to that of unmodified 2'-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite S-[2-(N6-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N6-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.

Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases.

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10-100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption.

Fate of triazoles in softwood upon environmental exposure.

Determining the fate of preservatives in commercial wood products is essential to minimize their losses and improve protective impregnation techniques. The fate of triazole fungicides in ponderosa pine wood was investigated in both outdoor and controlled-environment experiments using a representative triazole, tebuconazole (TAZ), which was accompanied by propiconazole (PAZ) in selected experiments. The study was designed to mimic industrial settings used in window frame manufacturing. To investigate the TAZ fate in detail, loosely and strongly bound fractions were differentiated using a multi-step extraction. The loosely bound TAZ fraction extracted through two sonications accounted for 85± 5% of the total TAZ, while the strongly bound TAZ was extracted only with an exhaustive Soxhlet extraction and corresponded to the remaining 15± 5%. A significant fraction (∼80%) of the original TAZ remained in the wood despite a six-month exposure to harsh environmental conditions, maintaining wood preservation and assuring minimal environmental impact. Depletion of loosely bound TAZ was observed from cross-sectional surfaces when exposed to rain, high humidity and sunlight. Water leaching was deemed to be the major route leading to triazole losses from wood. Leaching rate was found to be slightly higher for TAZ than for PAZ. The contribution of bio-, photo- and thermal degradation of triazoles was negligible as both PAZ and TAZ sorbed in wood remained intact. Triazole evaporation was also found to be minor at the moderate temperature (20-25 °C) recorded throughout the outdoor study.

Aromatic hydroxylation of salicylic acid and aspirin by human cytochromes P450.

Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.

Method development for the determination of wood preservatives in commercially treated wood using gas chromatography-mass spectrometry.

Fungicides and insecticides are commonly used preservatives to protect wood products against microbiological degradations. Currently, there is a lack of analytical methods addressing the quantitative determination of a wide range of wood preserving species in wood matrices. In this study, a reliable method was developed for the determination of a mixture of wood preserving agents with differing chemical structures (i.e., properties), including tebuconazole (TAZ), propiconazole (PAZ), 3-iodo-2-propynyl butylcarbamate (IPBC), and permethrin (PER), in pine wood. The analyte recoveries obtained by Soxhlet and multiple-stage sonication extractions were compared. While both extraction methods yielded similar results (80-100%), Soxhlet extraction was found to be less labor-intensive and thus preferred providing also lower RSDs of 1-6%. In comparison to methanol, commonly used as an extraction solvent for triazoles, acetone yielded similar extraction efficiencies for all analytes while reducing the time of sample concentration. The solid phase extraction method for triazoles was adapted to allow for a separation of IPBC and PER from the wood matrix. As opposed to previous studies, three recovery standards were employed, which enabled the correction of individual analyte losses during the sample preparation. The matrix-affected limits of detection (LODs) using gas chromatography with mass spectrometric detection were nearly the same for triazoles 0.07 and 0.21 ng g(-1) for PAZ and TAZ in sapwood and 0.18 and 0.21 ng g(-1) in heartwood, respectively. Higher LODs were observed for IPBC and PER: 3.9 and 1.7 ng g(-1) in sapwood, and 2.0 and 6.0 ng g(-1) in heartwood, respectively. The recoveries in the wood submitted to commercial sample treatment showed gradient distribution of analytes depending on the penetration of the treatment.