DNA looping by protamine follows a nonuniform spatial distribution

Abstract

DNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes proteins such as condensin. Here, however, we are interested in a different looping method whereby condensing agents (charge Rþ3) such as protamine proteins neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (%1 mm) DNA fragments using atomic force microscopy. We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine's in vivo role for looping DNA into toroids and the mechanism of DNA condensation by condensing agents more broadly.

The Art of Packaging the Sperm Genome: Molecular and Structural Basis of the Histone-To-Protamine Exchange

“…Recent studies using tethered particle motion assays and AFM found that salmon protamine uses a multi-step process, forming multiple independent loops of a roughly defined diameter that come together before forming a larger toroidal structure ( 137 ). Furthermore, the formation of these structures relies on protamine binding-and-bending the DNA, whereby multiple protamine molecules bind locally to a DNA segment to induce bending of the DNA filament to form loops ( 138 ). These data are in agreement with previous studies that identified loops formed by sperm DNA in vivo ( 119 , 121 , 124 ) as well as our recent EMSA and single molecule DNA curtain assays, which suggest that large-scale genome compaction by protamines is achieved by protamine protein cooperativity ( 95 ).…”

Protein-mediated DNA self-assembly by controlling the surface charge in a molecular crowding environment

“…14 Additionally, it was reported that chromosomal protein could help doublestranded DNA fold into loops. 15,16 These important pioneering works are intriguing and are excellent examples of artificial nanoarchitectures arising from DNA interactions with natural biomolecules. Nonetheless, most of the above-mentioned reports were proof-of-concept studies and limited to in vitro validation.…”