tailieunhanh - Báo cáo khoa học: Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1

The genome of the human pathogenEntamoeba histolytica, a primitive pro-tist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. | ễFEBS Journal Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1 Vijay Pal Yadav1 Prabhat Kumar Mandal Desirazu N. Rao3 and Sudha Bhattacharya1 1 Schoolof EnvironmentalSciences JawaharlalNehru University New Delhi India 2 Schoolof Life Sciences JawaharlalNehru University New Delhi India 3 Department of Biochemistry Indian Institute of Science Bangalore India Keywords EhLINE Entamoeba histolytica nicking endonuclease restriction endonuclease-like endonuclease RE-like endonuclease retrotransposon-encoded endonuclease Correspondence S. Bhattacharya Schoolof Environmental Sciences JawaharlalNehru University New Mehrauli Road New Delhi 110067 India Fax 91 11 26172438 Tel 91 11 26704308 E-mail sb@ sbjnu110@ Present address Department of Genetics University of Pennsylvania Schoolof Medicine Philadelphia PA USA Received 11 September 2009 revised 26 September 2009 accepted 30 September 2009 doi The genome of the human pathogen Entamoeba histolytica a primitive pro-tist contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction determined under steady-state and singleturnover conditions revealed a significant burst phase followed by a slower steady-state phase indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the Km was X 10 8 M and the kcat was X 10-2 sec-1. For linear E. histolytica DNA substrate the Km and kcat values were X 10-8 M and X 10-4 sec-1 respectively. Single-turnover reaction kinetics suggested a noncooperative mode of