Replication of G-quadruplex DNA by translesion polymerases

DNA复制对细胞来说是一项复杂而危险的工作，因为当双螺旋解开进行复制时，损害的风险最大。增加这种复杂性的是，一些DNA序列往往比其他序列更难准确复制。值得注意的是，这些忠实复制的天然障碍是采用非规范结构的序列基序，如G-四链(G4)DNA。未能保持进化上保守的G4基序会导致遗传和表观遗传不稳定，这与染色体损伤、细胞功能障碍、神经疾病和癌症有关。虽然人们知道需要特殊的处理来保持G4的完整性，但涉及的精确分子机制和细胞反应仍然是重要的研究领域。该项目将研究一种名为Rev1的聚合酶在复制和修复G4 DNA中的作用。该项目的成功完成将为绕过这种复制障碍提供新的知识，这是所有活着的系统的基本要求。该项目还将支持阿肯色大学医学科学(UAMS)的研究生培训、亨德里克斯学院的本科生培训，以及通过阿肯色州医科大学的外联计划对阿肯色州的高中生进行指导。该项目的主要目标是检验这一假说，即Rev1是一种具有不寻常作用机制的酶，其功能是破坏G4结构，将其他酶招募到G4诱导的复制应激部位，并在G4基序的复制过程中保持遗传/表观遗传的完整性。这项研究将使用生物物理和生化方法，包括瞬时动力学和蛋白质组学，以及基于细胞的分析，以研究Rev1活性的关键方面，这可能有助于G4复制和维持周围染色质的景观。这些成果将改进DNA复制模型，并提供对DNA加工途径的机械性见解，这些途径已经进化为维持真核生物基因组的重要结构特征。该项目由分子和细胞生物科学部和既定的刺激竞争研究计划(EPSCoR)联合资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

DNA replication is a complex and perilous undertaking for cells because the risk of damage is greatest when the double helix is unwound for copying. Adding to this complexity is the fact that some DNA sequences tend to be more difficult to replicate accurately than others. Notable among these natural barriers to faithful replication are sequence motifs that adopt non-canonical structures such as G-quadruplex (G4) DNA. Failure to maintain evolutionarily conserved G4 motifs leads to genetic and epigenetic instability that is associated with chromosomal damage, cellular dysfunction, neurological diseases, and cancer. While it is known that special processing is required to maintain G4 integrity, the precise molecular mechanisms and cellular responses involved remain important areas of research. This project will investigate the role of a polymerase called Rev1 in copying and repairing G4 DNA. Successful completion of the project will provide new knowledge on the bypass of such replication barriers, a fundamental requirement for all living systems. The project will also support graduate student training at the University of Arkansas for Medical Sciences (UAMS), undergraduate trainees at Hendrix College, and mentoring of high-school students in Arkansas through outreach programs housed at UAMS. The major goal of the project is to test the hypothesis that Rev1, an enzyme with an unusual mechanism of action, functions to disrupt G4 structures, recruit other enzymes to sites of G4-induced replication stress, and maintain genetic/epigenetic integrity during replication of G4 motifs. The research will employ biophysical and biochemical approaches, including transient kinetics and proteomics, as well as cell-based assays to study key aspects of Rev1 activity that likely facilitate G4 replication and maintenance of the surrounding chromatin landscape. The outcomes will improve models of DNA replication and provide mechanistic insights into DNA processing pathways that have evolved to maintain important structural features of eukaryotic genomes.This project is jointly funded by the Molecular and Cellular Biosciences Division and the Established Program to Stimulate Competitive Research (EPSCoR).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.