Generating Nucleosomal Ladders In Vivo by Releasing Endogenous Endonucleases in Chlamydomonas reinhardtii

Chromatin maps by enzymatic digestion using micrococcal nuclease (MNase) have served as popular substrates for molecular epigenetic studies. Such analyses have been limited to cell wall minus mutants of Chlamydomonas reinhardtii because of the complications in nucleus isolation due to cell wall removal. Here, we describe an endogenous endonuclease in Chlamydomonas that is specifically released from the cells upon abrasion with beads (glass or zirconia), in the presence of detergents. The resulting in vivo digests obtained from both cell wall containing cc124/cc125 and mutant strains (cc400) are typical of repetitive nucleosomal fragments (MNase-like ladders) ranging from mono- to poly-nucleosomes, indicating that the nuclease acts preferentially on inter-nucleosomal linker DNA. We demonstrate that the endonuclease activity is strictly dependent on divalent cations (Ca⁺²?>?Mg⁺²) and can be inhibited by both Cu⁺² and Zn⁺² (5 mM) or by divalent cation chelators like EDTA. Detailed standardization reveals that the nuclease is released only when the cells are treated with detergents, implying that it might be membrane bound or vesicular and intracellular in nature. This endonuclease seems to accumulate in late log-phase cultures and probably has a role in generation of apoptotic ladders in Chlamydomonas. Further activity-based in-gel DNase analyses of whole cell extracts, using denaturing SDS-PAGE, revealed a single major ~?30-kDa band upon renaturation. We further characterized this endonuclease by partial purification using ammonium sulfate precipitation. Activity-based analysis revealed partial enrichment in the 50% (NH₄)₂SO₄ peak fraction and also confirmed Ca dependence. Finally, adding back the released endonuclease from a detergent-treated supernatant of cc124 cells to intact cc400 cw ^? cells as substrate resulted in a typical ladder formation in the latter, confirming the intracellular release of the nuclease and its activity. In summary, by controlled release of calcium-dependent endogenous endonucleases (altering vortex and detergent conditions), we can generate dose-dependent nucleosomal ladders for epigenetic analyses in Chlamydomonas, as an alternate to MNase-derived substrates.