Department of Physics, Northeastern University, Boston, MA 02115, USA.
Department of Chemistry and Biochemistry, Center for Retroviral Research and Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA.
Nucleic Acids Res. 2023 Sep 8;51(16):8587-8605. doi: 10.1093/nar/gkad595.
Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32-ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32-ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.
噬菌体 T4 基因 32 蛋白（gp32）是一种模型单链 DNA（ssDNA）结合蛋白，对 DNA 复制至关重要。gp32 通过其核心和 N 端之间的蛋白间相互作用在 ssDNA 上形成协同纤维。然而，gp32 纤维结构和组织的详细理解仍然不完整，特别是对于更长的、与生物学相关的 DNA 长度。此外，尚不清楚这些紧密结合的纤维在互补链合成过程中如何从 ssDNA 上解离。我们使用光学镊子和原子力显微镜来探测长（约 8 knt）ssDNA 底物上 gp32 的结构和结合动力学。我们发现 gp32 的协同结合使 ssDNA 刚性化，同时也减少了其轮廓长度，这与 ssDNA 围绕 gp32 纤维螺旋缠绕一致。虽然测量的 gp32 结合和解离速率表明其具有 nM 的结合亲和力，但在约 1000 倍的更高蛋白浓度下，gp32 仍继续结合并重构 gp32-ssDNA 纤维，导致其螺旋螺距增加和底物伸长。此外，过饱和的 gp32-ssDNA 纤维变得越来越不稳定，并出现快速、非协同的蛋白质解离相，这在较低的复合物饱和度下没有观察到，这表明在复制过程中，聚合酶前方拥挤的 ssDNA 中 gp32 可能会迅速被去除的机制。
