PMID: 8995789Jul 1, 1996Paper

Evidence for a DNA homologous pairing activity in nuclear extracts from mosquito cells

Insect Biochemistry and Molecular Biology
G D Baldridge, A M Fallon

Abstract

Using a sensitive homologous pairing/DNA strand transfer assay, we detected formation of joint molecules in the presence of nuclear extract from cultured mosquito C7-10 cells in a reaction containing single stranded circular m13 DNA and a linear, double stranded DNA 5'-end-labeled on the strand complementary to a portion of the single-stranded substrate. Joint molecules were detected by the reduced electrophoretic mobility of labeled probe on agarose gels, which indicated that the 5'-end labeled strand of the linear duplex had formed a hybrid with the single-stranded substrate. Characterization of the activity detected initially in crude nuclear extracts provided a basis for a 5-fold enrichment of activity after a two-step KCl elution from heparin-Sepharose. Further purification by preparative electrophoresis yielded a band at approximately 35 kDa, which, when transferred to Immobilon P membrane, specifically bound the labeled, complementary strand probe. Optimal activity of the electroeluted enzyme required both magnesium and ATP and was sensitive to the ratio of single-stranded and double-stranded DNA substrate and to the amount of protein. This homologous pairing activity from mosquito cells is the first such activity to be ...Continue Reading

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