Development of protein purification methods for a putative sequence-specific endonuclease in Chlamydomonas

Abstract

Chlamydomonas reinhardtii is a key model organism for research into photosynthesis and other important cellular processes. A Chlamydomonas mutant library generated by the Jonikas Lab has been a powerful tool for genetic studies, though the process of mutant strain generation is inhibited by the activity of a putative sequence-specific DNA endonuclease. Sequence-specific DNA endonucleases have been well-studied in prokaryotic restriction-modification systems, yet few sequence-specific endonucleases have been identified and characterized in eukaryotic organisms. Here, I describe the development of protein purification protocols in order to identify this putative endonuclease in Chlamydomonas. The application of chromatography purification methods enriched upwards of 14 to 81-fold for the endonuclease, and a pilot mass spectrometry experiment that detected 1,317 enriched proteins suggests the multiplexing of these samples alongside negative control DNA pulldown samples will enable higher-confidence identification of the endonuclease. Further, preliminary data indicate the Ca2+ activation of the endonuclease alongside potential exonuclease activity. Altogether, this project builds towards improvements in mutant generation technologies and a greater understanding of the cell biology of sequence-specific endonucleases in eukaryotes.