DNA folding by protamine from loops to stacks.

Abstract

In sperm, DNA undergoes a dramatic reorganization to a semi-crystalline state. This incredible compaction is the work of positively charged protamine proteins which bend and loop negatively charged DNA into a series of toroids. Interestingly, other positively charged molecules such as spermine or cations such as cobalt (+3) have been shown to form these toroid structures in vitro, indicating that the physics that drives this process is likely to be electrostatic. Still, the exact mechanism and the states in the folding pathway are unknown. We have previously shown that multiple protamine molecules “bind and bend” the DNA into a loop. But, what happens after loop formation? To answer this question, we measured folding by protamine in vitro on DNA molecules (length = 600-3000 bp) that are known to form several loops. Using atomic force microscopy (AFM), we were able to identify several intermediate folded structures: molecules with a single loop (a loop), molecules with two or more loops that were connected at a single location like a flower (flowers), and molecules with two or more loops stacked on top of one another (stacks). Using a tethered particle motion (TPM) assay we measured real-time folding of the DNA into these structures. At low protamine concentrations (0.1 µM), we observed many, long-lived, discrete folded states indicative of protamine bending, but at higher concentrations (>0.1 µM) these states appeared as a single, initial folding event. We hypothesize that this single, initial folding event is protamine bending the DNA all along its length, causing multiple loops. These loops then collapse into a stack. More work is necessary to determine how these stacks eventually form toroids. This work is important for spermiogenesis and DNA condensation, but also may useful in DNA nanoengineering.