单链DNA结合蛋白(SSB)是细胞的重要组成部分,以高亲和力和序列非特异性结合到单链DNA(ssDNA)的瞬时暴露区域,以协调DNA修复和复制。大肠杆菌SSB(EcSSB)是一种同四聚体,将可变长度的ssDNA包裹在多种构象中(通常占据65或35nts),它在衬底长度的实验条件下得到了很好的研究,盐,pH值,温度,等。在这项工作中,我们使用原子力显微镜(AFM)来研究SSB与单个ssDNA分子的结合。我们引入了模拟自然发生的DNA损伤的非规范DNA碱基,合成无碱基位点,以及在预测与EcSSB相互作用的位点进入我们的实验构建体的非DNA接头。通过测量与EcSSB结合的DNA分子的分数以及每个DNA分子结合的蛋白质的体积,我们确定蛋白质结合亲和力,协同性,和构象。我们发现只有一个受损的核苷酸,EcSSB的结合相对于其与未受损DNA的结合是不变的。在存在两个串联脱碱基位点或非DNA间隔区的情况下,然而,在65nt模式下,与占据全部底物的单个EcSSB四聚体相关的结合亲和力大大降低。相比之下,两个EcSSB四聚体的结合,每个都在35nt模式下,被保存。EcSSB在这些构建体中的结合和合作行为的变化可以告知基因组修复和复制过程如何随着DNA中环境损伤的积累而变化。
Single-stranded DNA-binding proteins (SSBs) are essential cellular components, binding to transiently exposed regions of single-stranded DNA (ssDNA) with high affinity and sequence non-specificity to coordinate DNA repair and replication. Escherichia coli SSB (EcSSB) is a homotetramer that wraps variable lengths of ssDNA in multiple conformations (typically occupying either 65 or 35 nt), which is well studied across experimental conditions of substrate length, salt, pH, temperature, etc. In this work, we use atomic force microscopy to investigate the binding of SSB to individual ssDNA molecules. We introduce non-canonical DNA bases that mimic naturally occurring DNA damage, synthetic abasic sites, as well as a non-DNA linker into our experimental constructs at sites predicted to interact with EcSSB. By measuring the fraction of DNA molecules with EcSSB bound as well as the volume of protein bound per DNA molecule, we determine the protein binding affinity, cooperativity, and conformation. We find that, with only one damaged nucleotide, the binding of EcSSB is unchanged relative to its binding to undamaged DNA. In the presence of either two tandem abasic sites or a non-DNA spacer, however, the binding affinity associated with a single EcSSB tetramer occupying the full substrate in the 65-nt mode is greatly reduced. In contrast, the binding of two EcSSB tetramers, each in the 35-nt mode, is preserved. Changes in the binding and cooperative behaviors of EcSSB across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates in DNA.