Griffin Wezley C, Gao Jun, Byrd Alicia K, Chib Shubeena, Raney Kevin D
From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7101.
From the Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-7101
J Biol Chem. 2017 Jun 9;292(23):9567-9582. doi: 10.1074/jbc.M117.776211. Epub 2017 Apr 17.
DNA sequences that are guanine-rich have received considerable attention because of their potential to fold into a secondary, four-stranded DNA structure termed G-quadruplex (G4), which has been implicated in genomic instability and some human diseases. We have previously identified positive coactivator of transcription (PC4), a single-stranded DNA (ssDNA)-binding protein, as a novel G4 interactor. Here, to expand on these previous observations, we biochemically and biophysically characterized the interaction between PC4 and G4DNA. PC4 can bind alternative G4DNA topologies with a low nanomolar value of ∼2 nm, similar to that observed for ssDNA. In consideration of the different structural features between G4DNA and ssDNA, these binding data indicated that PC4 can interact with G4DNA in a manner distinct from ssDNA. The stoichiometry of the PC4-G4 complex was 1:1 for PC4 dimer:G4 substrate. PC4 did not enhance the rate of folding of G4DNA, and formation of the PC4-G4DNA complex did not result in unfolding of the G4DNA structure. We assembled a G4DNA structure flanked by duplex DNA. We find that PC4 can interact with this G4DNA, as well as the complementary C-rich strand. Molecular docking simulations and DNA footprinting experiments suggest a model where a PC4 dimer accommodates the DNA with one monomer on the G4 strand and the second monomer bound to the C-rich strand. Collectively, these data provide a novel mode of PC4 binding to a DNA secondary structure that remains within the framework of the model for binding to ssDNA. Additionally, consideration of the PC4-G4DNA interaction could provide insight into the biological functions of PC4, which remain incompletely understood.
富含鸟嘌呤的DNA序列因其具有折叠成称为G-四链体（G4）的二级四链DNA结构的潜力而备受关注，这种结构与基因组不稳定和一些人类疾病有关。我们之前已经鉴定出转录正向共激活因子（PC4），一种单链DNA（ssDNA）结合蛋白，是一种新型的G4相互作用蛋白。在此，为了扩展之前的这些观察结果，我们对PC4与G4DNA之间的相互作用进行了生物化学和生物物理表征。PC4可以以低纳摩尔亲和力（约2 nM）结合不同的G4DNA拓扑结构，这与观察到的其与ssDNA的结合情况相似。考虑到G4DNA和ssDNA之间不同的结构特征，这些结合数据表明PC4可以以不同于ssDNA的方式与G4DNA相互作用。PC4与G4复合物的化学计量比为PC4二聚体：G4底物 = 1:1。PC4不会提高G4DNA的折叠速率，并且PC4-G4DNA复合物的形成不会导致G4DNA结构的解折叠。我们组装了一个两侧为双链DNA的G4DNA结构。我们发现PC4可以与这种G4DNA以及互补的富含C的链相互作用。分子对接模拟和DNA足迹实验提出了一个模型，即PC4二聚体与DNA结合，其中一个单体位于G4链上，另一个单体与富含C的链结合。总的来说，这些数据提供了PC4与DNA二级结构结合的一种新模式，该模式仍在其与ssDNA结合模型的框架内。此外，考虑PC4-G4DNA相互作用可能有助于深入了解PC4尚未完全了解的生物学功能。
