tailieunhanh - Báo cáo khoa học: Chinese hamster apurinic⁄apyrimidinic endonuclease (chAPE1) expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation

Apurinic⁄apyrimidinic endonuclease (APE), an essential DNA repair enzyme, initiates the base excision repair pathway by creating a nick 5¢ to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies, the Chinese hamster APE (chAPE1) has not been studied in vitro in respect to its kinetic characteristics. | Chinese hamster apurinic apyrimidinic endonuclease chAPEI expressed in sf9 cells reveals that its endonuclease activity is regulated by phosphorylation Mandula Borjigin1 Bobbie Martinez2 Sarla Purohit2 Gaudalupe de la Rosa2 Pablo Arenaz2 and Boguslaw Stec3 1 Department of Chemistry Bowling Green State University OH USA 2 Department of BiologicalSciences Department of Chemistry University of Texas ElPaso USA 3 Sanford-Burnham MedicalResearch Institute La Jolla CA USA Keywords apurinic endonuclease caseine kinase phsphorylation DNA repair enzyme kinetics ICP regulation by phosphorylation Correspondence B. Stec Sanford-Burnham Medical Research Institute 10901 N. Torrey Pines Rd La Jolla CA 92037 USA Fax 858 795 5225 Tel 858 795 5257 E-mail bstec@ M. Borjigin Department of Chemistry 144 Overman Hall Bowling Green State University Bowling Green OH 43403 USA Fax 419 372 8088 Tel 419 372 8088 E-mail dman@ Received 7 June 2010 revised 30 August 2010 accepted 10 September 2010 doi Apurinic apyrimidinic endonuclease APE an essential DNA repair enzyme initiates the base excision repair pathway by creating a nick 5 to an abasic site in double-stranded DNA. Although the Chinese hamster ovary cells remain an important model for DNA repair studies the Chinese hamster APE chAPE1 has not been studied in vitro in respect to its kinetic characteristics. Here we report the results of a kinetic study performed on cloned and overexpressed enzyme in sf9 cells. The kinetic parameters were fully compatible with the broad range of kinetic parameters reported for the human enzyme. However the activity measures depended on the time point of the culture. We applied inductivity coupled plasma spectrometry to measure the phosphorylation level of chAPE1. Our data showed that a higher phosphorylation of chAPE1 in the expression host was correlated to a lower endonuclease activity. The phosphorylation of a higher activity batch of chAPE1 by casein