Fleming Aaron M, Jenkins Brandon Leonel Guerra Castañaza, Buck Bethany A, Burrows Cynthia J
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 United States.
bioRxiv. 2024 Jan 21:2024.01.20.576387. doi: 10.1101/2024.01.20.576387.
Molecular details for DNA damage impact on the folding of potential G-quadruplex sequences (PQS) to non-canonical DNA structures that are involved in gene regulation are poorly understood. Here, the effects of DNA base damage and strand breaks on PQS folding kinetics were studied in the context of the promoter sequence embedded between two DNA duplex anchors, referred to as a duplex-G-quadruplex-duplex (DGD) motif. This DGD scaffold imposes constraints on the PQS folding process that more closely mimic those found in genomic DNA. Folding kinetics were monitored by circular dichroism (CD) to find folding half-lives ranging from 2 s to 12 min depending on the DNA damage type and sequence position. The presence of Mg ions and the G-quadruplex (G4)-binding protein APE1 facilitated the folding reactions. A strand break placing all four G runs required for G4 formation on one side of the break accelerated the folding rate by >150-fold compared to the undamaged sequence. Combined 1D H-NMR and CD analyses confirmed that isothermal folding of the -DGD constructs yielded spectral signatures that suggest formation of G4 motifs, and demonstrated a folding dependency with the nature and location of DNA damage. Importantly, the PQS folding half-lives measured are relevant to replication, transcription, and DNA repair time frames.
DNA损伤对潜在的G-四链体序列（PQS）折叠成参与基因调控的非经典DNA结构的分子细节了解甚少。在此，在嵌入两个DNA双链锚之间的启动子序列（称为双链-G-四链体-双链（DGD）基序）的背景下，研究了DNA碱基损伤和链断裂对PQS折叠动力学的影响。这种DGD支架对PQS折叠过程施加了限制，更紧密地模拟了基因组DNA中的情况。通过圆二色性（CD）监测折叠动力学，发现折叠半衰期从2秒到12分钟不等，这取决于DNA损伤类型和序列位置。镁离子和G-四链体（G4）结合蛋白APE1的存在促进了折叠反应。与未受损序列相比，链断裂将形成G4所需的所有四个G序列置于断裂一侧，使折叠速率加快了150倍以上。结合一维H-NMR和CD分析证实，-DGD构建体的等温折叠产生了表明形成G4基序的光谱特征，并证明了折叠与DNA损伤的性质和位置有关。重要的是，测得的PQS折叠半衰期与复制、转录和DNA修复时间框架相关。
