Mechanism of completing cellular DNA replication

细胞完成染色体复制的机制还不清楚。 然而，这一过程必须非常有效，在人类细胞的每次细胞分裂中发生数千次，并且对于维持所有细胞生命和基因组稳定性至关重要。 这个项目将确定在细胞复制完成过程中发生的进行性分子中间体。 作为该项目的一部分，培训的学生将有资格从事医学，生物技术和研究工作，并将包括波特兰州立大学的毕业生和本科生，这是一所城市学院，学生人数超过40%是第一代大学生。该项目还将通过一系列活动加强波特兰公立学校系统的早期科学和数学教育，这些活动包括科学博览会、科学之夜、职业日、指导高中生研究以及学校发起的科学项目。完成复制涉及酶系统，该酶系统通过允许会聚的复制叉暂时绕过在过量的、过度复制的区域被切开、切除和接合之前彼此接合。 该反应通过SbcCD-ExoI的联合作用启动，并需要RecBCD连接复制叉的会聚链。 在完成复制受损的突变体中，细胞活力变得依赖于异常形式的重组，这也会导致遗传不稳定性。 虽然这些观察清楚地确立了这一基本过程的重要性，但这些酶催化反应的底物和机制仍然未知。本研究使用双向复制质粒微型染色体来鉴定在野生型细胞中发生的结构中间体和在体内完成反应受损的突变体。该项目将确定如何chi和ter序列，这是已知的调节完成酶，影响反应在体内使用基因组分析和质粒微型染色体。该研究还采用了基因筛选，旨在识别和表征完成过程中涉及的其他酶。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估。

The mechanism by which cells complete chromosome replication is not well understood. Yet the process must be remarkably efficient, occurring thousands of times per cell division in human cells, and is essential to maintain all cellular life and genome stability. This project will identify the processive molecular intermediates that occur during the completion of replication in cells. Students trained as part of this project will be qualified for careers in medicine, biotechnology, and research, and will include graduates and undergraduates at Portland State University, an urban college with a student body made up of more than 40% first generation college students. The project will also enhance early science and math education in the Portland public school system through a range of activities that include science fairs, science nights, career days, mentoring high school student research, and school-initiated science programs.In the bacterium Escherichia coli, completing replication involves an enzymatic system that limits cellular replication to its doubling point by allowing converging replication forks to transiently bypass each other before the excess, over-replicated regions are incised, resected, and joined. The reaction initiates through the combined action of SbcCD-ExoI and requires RecBCD to join convergent strands of the replication fork. In mutants where completing replication is impaired, cell viability becomes dependent on an aberrant form of recombination that also drives genetic instabilities. While these observations clearly establish the importance of this fundamental process, the substrates and mechanism by which these enzymes catalyze the reaction remain unknown. This research uses a bi-directionally replicating plasmid mini-chromosome to identify the structural intermediates that occur in wild type cells and mutants impaired for the completion reaction, in vivo. The project will determine how chi and ter sequences, which are known to regulate completion enzymes, affect the reaction in vivo using genomic profiling and plasmid mini-chromosomes. The research additionally employs a genetic screen designed to identify and characterize additional enzymes involved in the completion process.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.