Protamine loops DNA in multiple steps

Abstract

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops—c-shapes or s-shapes—that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.

DNA looping by protamine follows a nonuniform spatial distribution

“…Having confirmed that spontaneous loops are consistent with our random looping model, we next asked whether this model is also accurate for protamine-induced loops. Recently, we found that protamine-induced loops do not form in a single step (16). Instead, these loops form in multiple steps, with each step thought to correspond to one or more protamine molecules that bind the DNA and bend it into a particular radius of curvature (~10 nm) (16).…”

“…Recently, we found that protamine-induced loops do not form in a single step (16). Instead, these loops form in multiple steps, with each step thought to correspond to one or more protamine molecules that bind the DNA and bend it into a particular radius of curvature (~10 nm) (16). Multiple folding events, rather than just one event, are then needed to bend the DNA into a loop.…”

“…Some common examples of DNA condensing agents are cobalt (III) hexaammine (22,23), spermine (24)(25)(26), spermidine (26)(27)(28)(29), and protamine (16,17,30), although any cation with a charge of at least +3 is thought to function similarly (20), and some divalent cations have also been shown to condense DNA under certain conditions (20). DNA condensing agents are known to bind to DNA nonspecifically (11,20) and form loops (16,27,31), as well as toroids (20,32,33). Toroid sizes vary, but toroids generally have an outer diameter of about 100 nm (17,32) and can contain up to 50 kbp of DNA (30) in concentrically wound, hexagonally packed loops (22).…”

“…To fold the DNA into a loop, a previous model suggested that protamine stabilizes spontaneous loops (15,32). Recent evidence, however, suggests that protamine instead forms loops via a bind-and-bend mechanism (16) in which each protamine binds the DNA and induces a small (~20 ) bend.…”

“…There are different methods of DNA looping, each of which involves a different mechanism. One method of loop formation, spontaneous looping, occurs when thermal fluctuations cause two distal DNA segments to come together in the absence of proteins, creating a transient spontaneous loop (15,16). An experimental realization of spontaneous loops is when DNA is kinetically trapped on a surface (17).…”