Data from: Protamine folds DNA into flowers and loop stacks

DNA in sperm undergoes an extreme compaction to almost crystalline packing
levels. To produce this dense packing, DNA is dramatically reorganized in
minutes by protamine proteins. Protamines are positively charged proteins
that coat negatively charged DNA and fold it into a series of toroids. The
exact mechanism for forming these ∼50-kbp toroids is unknown. Our goal is
to study toroid formation by starting at the “bottom” with the folding of
short lengths of DNA that form loops and working “up” to more folded
structures that occur on longer length scales. We previously measured the
folding of 200–300 bp of DNA into a loop. Here, we look at
folding of intermediate DNA lengths (L = 639–3003 bp)
that are 2–10 loops long. We observe two folded structures besides loops
that we hypothesize are early intermediates in the toroid formation
pathway. At low protamine concentrations (∼0.2 μM), we see that the DNA
folds into flowers (structures with multiple loops that are positioned so
they look like the petals of a flower). Folding at these concentrations
condenses the DNA to 25% of its original length, takes seconds, and is
made up of many small bending steps. At higher protamine concentrations
(≥2 μM), we observe a second folded structure—the loop stack—where loops
are stacked vertically one on top of another. These results lead us to
propose a two-step process for folding at this length scale: 1) protamine
binds to DNA, bending it into loops and flowers, and 2) flowers collapse
into loop stacks. These results highlight how protamine uses a
bind-and-bend mechanism to rapidly fold DNA, which may be why protamine
can fold the entire sperm genome in minutes.

精子内的DNA会经历极致压缩，达到近乎类晶体的包装水平。为实现这种致密包装，鱼精蛋白（protamine）可在数分钟内对DNA进行大幅重组。鱼精蛋白是一类带正电荷的蛋白质，能够结合带负电荷的DNA，并将其折叠为一系列环形结构（toroid）。目前，这类尺寸约为50千碱基对（kbp）的环形结构的确切形成机制仍不明确。本研究旨在从‘基础层面’入手研究环形结构的形成过程：先解析形成DNA环（loop）的短片段DNA的折叠机制，再逐步拓展至更长长度尺度下的高级折叠结构。此前，我们已测得200~300碱基对（bp）的DNA可折叠为DNA环。本文中，我们将研究长度介于639~3003碱基对、对应2~10个DNA环的中等长度DNA的折叠行为。我们观测到除DNA环之外的两种折叠结构，并假设它们是环形结构形成通路中的早期中间产物。在低浓度鱼精蛋白（约0.2 μM）条件下，DNA会折叠为‘花状结构（flowers）’——即多个DNA环按类似花瓣的排布方式形成的复合体。该折叠过程可将DNA压缩至原长的25%，耗时仅数秒，且由诸多微小的弯曲步骤构成。在高浓度鱼精蛋白（≥2 μM）条件下，我们观测到第二种折叠结构——‘环堆叠结构（loop stack）’，即多个DNA环沿垂直方向层层堆叠。基于上述结果，我们提出该长度尺度下的DNA折叠分为两个步骤：1）鱼精蛋白结合DNA，将其弯曲形成DNA环与花状结构；2）花状结构坍塌为环堆叠结构。本研究结果揭示了鱼精蛋白如何通过‘结合-弯曲’机制快速折叠DNA，这或许正是鱼精蛋白能够在数分钟内完成整个精子基因组折叠的原因。