• Prolay Das

      Articles written in Journal of Biosciences

    • Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation

      Prolay Das Paula Bennett Betsy M Sutherland

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      Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10–100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low-LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.

    • Accelerated processing of solitary and clustered abasic site DNA damage lesions by APE1 in the presence of aqueous extract of Ganoderma lucidum

      Bhavini Kumari Prolay Das Rekha Kumari

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      The stimulatory effect of the aqueous extract of G. lucidum, a basidiomycetes class fungus in the APE1-enzyme-mediated processing of solitary and bistranded clustered abasic sites DNA damages is presented. Abasic sites are considered the most common type of DNA damage lesions. Our study shows enhanced activity of APE1 in the processing of abasic sites in the presence of the polysaccharides fraction of G. lucidum. Remarkable increase in the amount of single-strand breaks (SSBs) and double-strand breaks (DSBs) from solitary and bistranded clustered abasic sites respectively with APE1 in the presence of the extract was found. This trend is maintained when abasic sites in DNA oligomers are exposed to fibroblast cell extracts in the presence of the extract. While DNA conformational alteration is negligible, APE1 enzyme shows characteristic changes in the alpha helix and beta strand ratio after incubation with G. lucidum extract. The enhanced reactivity of APE1 at the molecular level in the presence of G. lucidium is attributed to this effect. This study potentially amplifies the scope of the use of G. lucidum, which was earlier shown to have only reactive oxygen species (ROS) scavenging properties with regards to DNA damage inhibition.

    • Complex interplay of lesion-specific DNA repair enzyme on bistranded clustered DNA damage harboring Tg:G mismatch in nucleosome core particles


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      5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) and 7,8-dihydro-8-oxo-20-deoxyguanosine (8-oxodG) are major DNAdamage lesions produced by endogenous oxidative stress, as well as inflicted by carcinogens and ionizing radiation. Theprocessing of Tg:G mismatch and 8-oxodG in close proximity of each other in a bistranded clustered environment in DNAoligomer duplexes as well as in a nucleosome core particle (NCP) model are reported here. The processing of the lesionswas evaluated by purified enzyme cocktails of hNTH1 and hOGG1 as well as with a HeLa cell extract. Interestingly, theyield of double-strand breaks (DSBs) resulting from the processing of the bistranded lesions are appreciably lower when theDNA is treated with the HeLa cell extract compared with the relevant purified enzyme cocktail in both models. Clusteredbistranded lesions become more repair refractive when reconstituted as an NCP. This indicates a complex interplay betweenthe repair enzymes that influence the processing of the bistranded cluster damage positively to avoid the formation of DSBsunder cellular conditions. In addition to position and orientation of the lesions, the type of the lesions in the clusterenvironment in DNA along with the relative abundance of the lesion-specific enzymes in the cells strongly prevents theprocessing of the oxidized nucleobases.

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