Excision Repair of Adducted DNA by Extracts from C. pyrenoidosa

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Project title

Code/計畫編號

NSC88-2313-B019-048

Translated Name/計畫中文名

單胞藻對DNA外接鍵結物之切割修補作用

Project Coordinator/計畫主持人

Todd Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=427528

Year

1999

Start date/計畫起

01-08-1998

Expected Completion/計畫迄

01-07-1999

Bugetid/研究經費

591千元

ResearchField/研究領域

生物科學

以抗癌藥劑Cisplatin處理過之質體DNA為體外DNA修補試驗之基質證實小球單胞藻抽取液含有核酸切割修補作用蛋白質。單胞藻抽取液可使受Cisplatin(16.8 pmole)處理之質體DNA(1.mu.g)增加9倍之修補合成作用。當固化在agarose顆粒上之紫外線傷害DNA與單胞藻抽取液在30.degree.C培養20分鐘後,此抽取液幾乎喪失切割修補Cisplatin所造成DNA損害之能力,然而與未照射固化DNA培養之抽取液則有非常明顯之修補活性。此一現象顯示固化後之紫外線傷害DNA可有效吸附小球單胞藻抽取液中之DNA修補蛋白質。SDS-PAGE蛋白電泳分析吸附後之蛋白質鮮示有一分子量介於70至72KDa者對紫外線傷害DNA有明顯親和力,表示此一蛋白質極有可能參與小球單胞藻之核酸切割修補作用。An in vitro DNA repair synthesis assay monitoring nucleotide excision repair (NER) was established in cell-free extracts of unicellular alga Chlorella pyrenoidosa using cisplatin-damaged plasmid as the repair substrate. The ratio of cisplatin (16.8 pmole) -induced to control dAMP incorporation was estimated to be 9.0 in several experiments. A biotin-labeled 27 mer (2.mu.g) was irradiated with UV (27KJ/m/sup 2/) and immobilized on streptavidin-conjugated agarose beads, and this insolubilized DNA was incubated at 30.degree.C for 20 min with algal extracts to pull down NER factors by affinity adsorption. The extracts post incubation with the beads carrying unirradiated or no DNA ligand showed a repair factor between 8 and 9 for cisplatin-damaged DNA, whereas the extracts incubated with the beads carrying UV-damaged DNA almost lost NER capacity, reflecting that some factors crucial to NER had been captured by UV-damaged DNA immobilized on the beads. SDS-PAGE analysis of the captured proteins clearly indicated the preferential binding of a polypeptide about 70 to 72KDa to UV-irradiated DNA. The binding of polypeptide p72 to control DNA was barely detectable, and this polypeptide was therefore highly speculated to play a role in NER in C.pyrenoidosa.

Keyword(s)

單胞藻
DNA修復
核酸修補
紫外線
順氯氨帕
Chlorella pyrenoidosa
DNA repair
Nucleotide excision repair
Ultraviolet light
Cisplatin

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Excision Repair of Adducted DNA by Extracts from C. pyrenoidosa

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