Abstract：
Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32\'s C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing.
摘要：
噬菌体T4基因32蛋白(gp32)是DNA复制所必需的单链DNA(ssDNA)结合蛋白。gp32通过其核心和N末端结构域之间的协同相互作用在ssDNA上形成稳定的蛋白质丝。gp32的C端结构域（CTD）被认为主要通过与复制体成分的直接相互作用来帮助协调DNA复制。然而,这些相互作用的确切机制尚不清楚，尚不清楚紧密结合的gp32细丝如何容易从ssDNA中取代，这是基因组加工所需的。这里,我们利用截短的gp32变体来证明CTD在调节gp32解离中的关键作用。使用光学镊子，我们探测了CTD截短的gp32，*I的结合和解离动力学，与8.1kntssDNA分子进行比较，并将这些测量值与全长gp32的测量值进行比较。*I-ssDNA螺旋丝随着蛋白质浓度的增加而逐渐解开，但仍然比全长的更稳定，野生型gp32。蛋白质过饱和,伴随着长丝退绕，促进全长gp32从整个ssDNA片段的快速解离。相比之下,*我主要从合作集群的末端缓慢解除绑定，无论蛋白质密度和DNA解链程度如何。我们的结果表明，CTD可能会限制ssDNA丝内蛋白质的相对扭曲角，因此在关键解链时，蛋白质间的协同相互作用将大大消失，便于及时删除gp32。我们提出了一个CTD介导的gp32位移模型，通过其细丝的内部重组，提供了在基因组加工过程中快速清除ssDNA的机制。
