Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.     

Linear dichroism studies of conformations of carcinogen-DNA adducts application to covalent complexes derived from the reactions of the two enantiomers of 9,10-epoxy-9,10,11,12-tetrahydrobenzo(e)pyrene with DNA

The conformations of the adducts derived from the covalent binding of the two enantiomeric forms of 9,10-epoxy-9,10,11,12-tetrahydrobenzo(e)pyrene (BePE) with native DNA were investigated by the electric linear dichroism technique. Both enantiomers give rise to two major adducts, one of which appears to be a quasi-intercalative site (I) while the other one is an external binding site (II). While the overall linear dichroism spectra are similar, in the case of the (-) enantiomer there is a greater contribution of site II adducts. These results are markedly different from the ones obtained with the two enantiomers of anti-benzo(a)pyrene-7,8-diol-9,10-epoxide (BaPDE), where the (+) enantiomer gives rise almost exclusively to site II binding, while the (-) enantiomer gives rise to both site I and site II covalent binding. The differences in the heterogeneity of binding between BePE and anti-BaPDE enantiomers may be due to the absence of hydroxyl groups in BePE which, in the case of BaPDE, are an important factor in determining the stereoselective properties of the covalent binding to double-stranded DNA.     

Bimolecular quenching of excitons and fluorescence in the photosynthetic unit.

The recent results of Campillo et al. and Mauzerall on the quenching of the fluorescence of chlorophyll a in Chlorella pyrenoidosa as a function of the intensity of the laser excitation pulses are rationalized by applying a model invoking singlet-singlet exciton annihilation.     

黑龙江东部双鸭山、集贤煤田中生代含煤地层研究*

本文系统地讨论了黑龙江双鸭山、集贤两煤田的中生代含煤地层序列,综合各门类化石研究结果,认为海相的绥滨组和东荣组属晚侏罗世卡洛夫晚期至伏尔加期,非海相的城子河组和穆棱组属早白垩世尼坎期。文内提供11条详细的钻探剖面的生物地层资料。     

Exciton Annihilation in the Two Photosystems in Chloroplasts at 100°K

The fluorescence yield (F) of spinach chloroplasts at 100°K measured at 735 nm (photosystem I fluorescence—F 735) and at 685 nm (photosystem II fluorescence—F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-μs pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10¹⁵ to 10¹⁸ photons/cm² per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.     

Stereochemistry-dependent bending in oligonucleotide duplexes induced by site-specific covalent benzo[a]pyrene diol epoxide-guanine lesions.

The apparent persistence length of enzymatically linearized pIBI30 plasmid DNA molecules approximately 2300 bp long, as measured by a hydrodynamic linear flow dichroism method, is markedly decreased after covalent binding of the highly tumorigenic benzo[a]pyrene metabolite 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. In striking contrast, the binding of the non-tumorigenic, mirror-image 7S,8R,9R,10S enantiomer [(-)-anti-BPDE] to DNA has no measurable effect on its alignment in hydrodynamic flow gradients (< or = 2.2% of the DNA bases modified). In order to relate this effect to BPDE-nucleotide lesions of defined stereochemistry, the bending induced by site-specifically placed and stereochemically defined (+)- and (-)-anti-BPDE-N2-dG lesions in an 11mer deoxyoligonucleotide duplex was studied by ligation and gel electrophoresis methods. Out of the four stereochemically isomeric anti-BPDE-N2-deoxyguanosyl (dG) adducts with either (+)-trans, (-)-trans, (+)-cis, and (-)-cis adduct stereochemistry, only the (+)-trans adduct gives rise to prominent bends or flexible hinge joints in the modified oligonucleotide duplexes. Since both anti-BPDE enantiomers are known to bind preferentially to dG (> or = 85%), these observations can account for the differences in persistence lengths of DNA modified with either (+)-anti-BPDE or the chiral (-)-anti-BPDE isomer.     

Costly sexual harassment in a beetle

Abstract The optimal number of mating partners for females rarely coincides with that for males, leading to sexual conflict over mating frequency. In the bruchid beetle Callosobruchus maculatus, the fitness consequences to females of engaging in multiple copulations are complex, with studies demonstrating both costs and benefits to multiple mating. However, females kept continuously with males have a lower lifetime egg production compared with females mated only once and then isolated from males. This reduction in fitness may be a result of damage caused by male genitalia, which bear spines that puncture the female’s reproductive tract, and/or toxic elements in the ejaculate. However, male harassment rather than costs of matings themselves could also explain the results. In the present study, the fitness costs of male harassment for female C. maculatus are estimated. The natural refractory period of females immediately after their first mating is used to separate the cost of harassment from the cost of mating. Male harassment results in females laying fewer eggs and this results in a tendency to produce fewer offspring. The results are discussed in the context of mate choice and sexual selection.     

Two-step error-prone bypass of the (+)- and (−)-trans-anti-BPDE-N-dG adducts by human DNA polymerases η and κ

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase η (Polη) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Polη predominantly inserted an A opposite a template (+)- and (−)-trans-anti-BPDE-N²-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Polη. Error-prone nucleotide insertion by human Polη was more efficient opposite the (+)-trans-anti-BPDE-N²-dG adduct than opposite the (−)-trans-anti-BPDE-N²-dG. However, translesion synthesis by human Polη largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Polη from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N²-dG adducts, the nucleotide opposite the lesion, and the sequence context 5′ to the lesion. By combining the nucleotide insertion activity of human Polη and the extension synthesis activity of human Polκ, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (−)-trans-anti-BPDE-N²-dG DNA adducts.