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To assess how evolution might have biochemically shaped DNA polymerase 1/ (Polt/) in plants. we expressed in E.scherichia co/i proteins from .4rabidopsis thu/lana (At), humans (Hs). and the yeast Saecharomycea cereuisiae (Sc), purified them to near homogeneity, and compared their properties. Consistent with the multiple divergent amino acids within mostly conserved polymerase domains, the polymerases showed modest, appreciable, and marked differences. respectively, in salt and temperature optima for activity and thermostability. We compared abilities to extend synthetic primers past template cyclobutane thymine dimers (T[CPD]T) or undamaged T-T under physiological conditions (80-I It) mM salt). Specific activities for "standing-start" extension of synthetic primers ending opposite the second template nucleotide 3' to T-T were roughly similar. DUriTig subsequent "running-start" insertions past T-T and the next 5' (N + I) nucleotide, AtPolt7 and HsPolij appeared more processive. but DNA sequence contexts strongly affected termination probabilities. Lesion-bypass studies employed four different templates containing TICPD]Ts, and two containing pyrimidine (6--t')-pyrimidinone photoproducts (16-415). AtPol~ made the three successive insertions [opposite the TICPD]T and (N + I) nucleotidesi that define bypass nearly as well as HsPoltj and somewhat better than ScPol~. Again, sequence context elfects were profound. Interestingly, the level of insertion opposite the (N - I) nucleotide 3' to TICPD1T by HsPolt/ and especially AtPolsi, but not ScPolli. was reduced (up to 4-fold) relative to the level of insertion opposite the (N - I) nucleotide 3' to T-T. Evolutionary conservation of efficient T[CPD]T bypass by HsPolq and AtPols~ may reflect a high degree of exposure of human skin and plants to solar UV-B radiation. The depressed (N - I) insertion upstream of T[CPDIT (but not T-T) may reduce the extent of gratuitous error-prone insertion. [ABSTRACT FROM AUTHOR]

Abstract: Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2·MSH6 (MutSα), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5–30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3′–5′ excision at the nicks. We compared these results to binding of purified MutSα to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSα binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSα binding affinities, suggesting that high hMutSα binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways. [Copyright &y& Elsevier]

Abstract: Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins—bacterial MutS, eukaryotic MSH2·MSH6 (MutSα) and MSH2·MSH3–initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of “mismatched” T[CPD]T/AG and T[6–4]T/AG photoproducts, reported previously for hMutSα and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6–4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K 1/2 2.1–2.9nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6–4]T/AG a forked H-bond between O2 of the 3′ thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6–4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSα) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis. [Copyright &y& Elsevier]

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity. [ABSTRACT FROM AUTHOR]

Strong frequency dependence of particle aggregation in applied ac-electric fields is reported for colloidal assembly under positive dielectrophoresis. A curious low-frequency threshold ωth on the order of 104–105 Hz is observed for the onset of collecting submicron latex particles at the high-field regions near coplanar electrodes. Similar to the dielectrophoretic crossover frequency, ωth is independent of medium conductivity, σm at low-salt concentration, yet scales with σm0.5 at high-salt concentrations. This intriguing salt effect suggests the distinct contribution from Stern and diffuse layer conductance to the interfacial polarization mechanism of latex particles in nonuniform ac fields. [ABSTRACT FROM AUTHOR]

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the β-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C → T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C → A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT → TC and TC → TT reversions were both high. With increased transgene copy number, spontaneous G:C → T:A reversions increased-but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta. [ABSTRACT FROM AUTHOR]

Cell survival after DNA damage depends on specialized DNA polymerases able to perform DNA synthesis on imperfect templates. Most of these enzymes belong to the recently discovered Y-family of DNA polymerases, none of which has been previously described in plants. We report here the isolation, functional characterization and expression analysis of a plant representative of the Y-family. This polymerase, which we have termed AtPolκ, is a homolog of Escherichia coli pol IV and human pol κ, and thus belongs to the DinB subfamily. We purified AtPolκ and found a template-directed DNA polymerase, endowed with limited processivity that is able to extend primer–terminal mispairs. The activity and processivity of AtPolκ are enhanced markedly upon deletion of 193 amino acids (aa) from its carboxy (C)-terminal domain. Loss of this region also affects the nucleotide selectivity of the enzyme, leading to the incorporation of both dCTP and dTTP opposite A in the template. We detected three cDNA forms, which result from the alternative splicing of AtPOLK mRNA and have distinct patterns of expression in different plant organs. Histochemical localization of β-glucuronidase (GUS) activity in transgenic plants revealed that the AtPOLK promoter is active in endoreduplicating cells, suggesting a possible role during consecutive DNA replication cycles in the absence of mitosis. [ABSTRACT FROM AUTHOR]

We describe modified gas chromatography electron-impact/triple-quadrupole mass spectrometry (GC–EI/MS/MS) utilizing a newly developed hydrogen-injected self-cleaning ion source and modified 9 mm extractor lens. This instrument, with optimized parameters, achieves quantitative separation of 62 polycyclic aromatic hydrocarbons (PAHs). Existing methods historically limited rigorous identification and quantification to a small subset, such as the 16 PAHs the US EPA has defined as priority pollutants. Without the critical source and extractor lens modifications, the off-the-shelf GC–EI/MS/MS system was unsuitable for complex PAH analysis. Separations were enhanced by increased gas flow, a complex GC temperature profile incorporating multiple isothermal periods, specific ramp rates, and a PAH-optimized column. Typical determinations with our refined GC–EI/MS/MS have a large linear range of 1–10,000 pg μl −1 and detection limits of <2 pg μl −1 . Included in the 62 PAHs, multiple-reaction-monitoring (MRM) mode enabled GC-EI/MS/MS identification and quantitation of several constituents of the MW 302 PAH isomers. Using calibration standards, values determined were within 5% of true values over many months. Standard curve r 2 values were typically >0.998, exceptional for compounds which are archetypally difficult. With this method benzo[a]fluorene, benzo[b]fluorene, benzo[c]fluorene were fully separated as was benzo[b]fluoranthene, benzo[k]fluoranthene, and benzo[j]fluoranthene. Chrysene and triphenylene, were sufficiently separated to allow accurate quantitation. Mean limits of detection (LODs) across all PAHs were 1.02 ± 0.84 pg μl −1 with indeno[1,2,3-c,d] pyrene having the lowest LOD at 0.26 pg μl −1 and only two analytes above 2.0 pg μl −1 ; acenaphthalene (2.33 pg μl −1 ) and dibenzo[a,e]pyrene (6.44 pg μl −1 ). [ABSTRACT FROM AUTHOR]

[Display omitted] Studies suggest that exposure to potential endocrine disrupting chemicals (pEDCs) may contribute to adverse health outcomes, but pEDC exposures among firefighters have not been fully characterized. Previously, we demonstrated the military-style silicone dog tag as a personal passive sampling device for assessing polycyclic aromatic hydrocarbon exposures among structural firefighters. This follow-up analysis examined the pEDC exposures based on department call volume, duty shift, and questionnaire variables. Structural firefighters (n = 56) were from one high and one low fire call volume department (Kansas City, MO metropolitan area) and wore separate dog tags while on- and off-duty (n dogtags = 110). The targeted 1530 analyte semi-quantitative screening method was conducted using gas chromatography mass spectrometry (n pEDCs = 433). A total of 47 pEDCs were detected, and several less-frequently-detected pEDCs (<75%) were more commonly detected in off- compared to on-duty dog tags (conditional logistic regression). Of the 11 phthalates and fragrances detected most frequently (>75%), off-duty pEDC concentrations were strongly correlated (r = 0.31–0.82, p < 0.05), suggesting co-applications of phthalates and fragrances in consumer products. Questionnaire variables of "regular use of conventional cleaning products" and "fireplace in the home" were associated with select elevated pEDC concentrations by duty shift (paired t -test). This suggested researchers should include detailed questions about consumer product use and home environment when examining personal pEDC exposures. [ABSTRACT FROM AUTHOR]

• Silicone dog tags are a new configuration of personalized passive sampling device. • Silicone dog tags detected 18 PAHs not previously reported as firefighter exposures. • Firefighters have higher chemical exposures on– compared to off-duty. • Concentrations were more correlated to fire attack numbers than firefighter rank. • Silicone dog tags can provide quantitative exposure data on chemical mixtures. Occupational chemical hazards in the fire service are hypothesized to play a role in increased cancer risk, and reliable sampling technologies are necessary for conducting firefighter chemical exposure assessments. This study presents the military-style dog tag as a new configuration of silicone passive sampling device to sample individual firefighters' exposures at one high and one low fire call volume department in the Kansas City, Missouri metropolitan area. The recruited firefighters (n = 56) wore separate dog tags to assess on– and off-duty exposures (n dogtags = 110), for a total of 30 24 h shifts. Using a 63 PAH method (GC–MS/MS), the tags detected 45 unique PAHs, of which 18 have not been previously reported as firefighting exposures. PAH concentrations were higher for on– compared to off-duty tags (0.25 < Cohen's d ≤ 0.80) and for the high compared to the low fire call volume department (0.25 ≤ d < 0.70). Using a 1530 analyte screening method (GC–MS), di-n-butyl phthalate, diisobutyl phthalate, guaiacol, and DEET were commonly detected analytes. The number of fire attacks a firefighter participated in was more strongly correlated with PAH concentrations than firefighter rank or years in the fire service. This suggested that quantitative data should be employed for firefighter exposure assessments, rather than surrogate measures. Because several detected analytes are listed as possible carcinogens, future firefighter exposure studies should consider evaluating complex mixtures to assess individual health risks. [ABSTRACT FROM AUTHOR]