Human DNA polymerase β initiates DNA synthesis during long-patch repair of reduced AP sites in DNA

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase β (Pol β) is responsible for the repair synthesis in this pathway and also removes a 5′-sugar phosphate residue by catalyzing a β-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to β-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol β, Pol δ and Pol ε) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to β-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol β is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis

Testing the Oxidative Stress Hypothesis of Aging in Primate Fibroblasts: Is There a Correlation Between Species Longevity and Cellular ROS Production?

The present study was conducted to test predictions of the oxidative stress theory of aging assessing reactive oxygen species production and oxidative stress resistance in cultured fibroblasts from 13 primate species ranging in body size from 0.25 to 120 kg and in longevity from 20 to 90 years. We assessed both basal and stress-induced reactive oxygen species production in fibroblasts from five great apes (human, chimpanzee, bonobo, gorilla, and orangutan), four Old World monkeys (baboon, rhesus and crested black macaques, and patas monkey), three New World monkeys (common marmoset, red-bellied tamarin, and woolly monkey), and one lemur (ring-tailed lemur). Measurements of cellular MitoSox fluorescence, an indicator of mitochondrial superoxide (O2(·-)) generation, showed an inverse correlation between longevity and steady state or metabolic stress-induced mitochondrial O2(·-) production, but this correlation was lost when the effects of body mass were removed, and the data were analyzed using phylogenetically independent contrasts. Fibroblasts from longer-lived primate species also exhibited superior resistance to H(2)O(2)-induced apoptotic cell death than cells from shorter-living primates. After correction for body mass and lack of phylogenetic independence, this correlation, although still discernible, fell short of significance by regression analysis. Thus, increased longevity in this sample of primates is not causally associated with low cellular reactive oxygen species generation, but further studies are warranted to test the association between increased cellular resistance to oxidative stressor and primate longevity

VALIDATION OF THE HUMAN T-LYMPHOCYTE CLONING ASSAY--RING TEST REPORT FROM THE EU CONCERTED ACTION ON HPRT MUTATION

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P = 0.0004) and lnMF (P = 0.03), but there was no significant laboratory effect on the lnCE (P = 0.38) or lnMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations

Radio Sensitivity of the winter and spring barley varieties according to the morphological efect of low-dose gamma irradiation on the original seeds.

Barley is the main forage crop in the Russian Federation. Barley grain is also used for food and brewery (Filippov, 2013). It is common knowledge that low doses of gamma irradiation can have a stimulating effect on the growth and development of plants, in particular barley. However, there is a lack of knowledge about the molecular pathways responsible for the formation of the stimulation effect after low-dose seed irradiation. The use of varieties with different radio sensitivity to low-dose gamma irradiation will allow studying specific molecular mechanisms to form a stimulating irradiation effect. Such knowledge could further help in the development of varieties with a large stable productivity over the years and with high resistance to biotic and abiotic stressors. In 2019 there was conducted a gamma irradiation of the original seeds of nine winter and spring barley varieties developed in the FSBSI “Agricultural Research Center “Donskoy” in order to assess intensity of the response on low-dose ionizing irradiation based on changes in the morphological parameters of irradiated and unirradiated plants. The original seeds of each barley variety were irradiated at the FSBSI “All-Russian Research Institute of Radiology and Agroecology” by the gamma device “GUR-120” with 60Со radiation sources at a dose of 20 Gy (dose rate of 60 Gy/hour). The statistical data processing was performed by the Microsoft Office Excel 2019. The morphological analysis of the length and weight of roots and sprouts made it possible to establish the presence or absence of the stimulating irradiation effect on the studied indicators of the varieties and to conduct their subsequent ranking according to the value of the radiobiological effect. There have been identified the winter and spring barley varieties with different sensitivity to a dose of 20 Gy. The stimulating irradiation effect was established in such varieties as “Foks 1”, “Ratnik”, “Yerema” and “Master”. The variety “Leon” had an inhibitory irradiation effect