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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/37642

Title: Purification of a UV-damaged-DNA binding activity from cell-free extracts of unicellular alga Chlorella pyrenoidosa
Authors: Todd Hsu;Jhon-Chun Ho;Chih-Chung Chao
Contributors: 國立台灣海洋大學:生命科學系
Keywords: Alga;Binding protein;Chlorella pyrenoidosa;Nucleotide excision repair;Ultraviolet light
Date: 1998-11
Issue Date: 2016-04-07T08:02:23Z
Publisher: Plant Science
Abstract: Abstract:Several binding activities recognizing ultraviolet (UV)-induced DNA damage were detected in cell-free extracts of the unicellular alga, Chlorella pyrenoidosa by gel shift assay. A UV-damaged-DNA binding activity was isolated from algal extracts by a series of chromatography on diethylaminoethyl-cellulose (DEAE-cellulose) and heparin-sepharose columns. The binding activity was detected in the flowthrough (FT) of heparin affinity chromatography. Quantitative analysis of the shifted bands after gel shift assay indicated that the ratio of UV-specific (18 kJ m−2) to non-specific binding increased about 15-fold from the first DEAE fractionation to heparin affinity chromatography. When the heparin-FT was fractionated again on a DEAE-cellulose column, UV-dependent binding was detected only in the fraction eluted by NaCl between 0.20 and 0.21 M. Three polypeptides in this NaCl eluate, estimated to be 72, 80 and 90 kDa in molecular mass by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), were shown to bind directly to UV-damaged DNA. This binding activity, however, showed only 25% of the binding affinity provided by the heparin-FT, implying that some modulating factors for UV-dependent binding might exist in the heparin-FT. Thus, the three polypeptides may represent a core UV-damaged-DNA binding activity in algal extracts. This core binding activity also recognized DNA lesions induced by DNA alkylating agent cisplatin, and the binding to cisplatin-damaged DNA could be competed by UV-irradiated DNA. Our data suggest that this binding activity may be involved in the damage-recognition step of nucleotide excision repair (NER) in C. pyrenoidosa.
Relation: 138(2),pp.137-147
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/37642
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