The Completion of DNA Replication

染色体复制准确完成的机制尚不清楚。为了在复制过程中保持基因组的完整性，细胞必须具有识别复制区域的能力，限制正在进行的复制过程，并在DNA序列翻倍的精确点连接聚合子链。尽管存在这些复杂性，但这一过程以惊人的效率发生（在人类细胞的每次分裂中发生数千次），并且是精确复制基因组的基础。在许多癌细胞中，它似乎是一个共同的解除管制的点，并且被病毒故意以灭活为目标，以诱导复制的裂解阶段（在此阶段病毒迅速繁殖）。该项目将确定细胞如何感知复制区域，并在模式生物（大肠杆菌和酿酒酵母）的精确加倍点准确完成这一过程。在此项目中接受培训的学生将有资格在学术界和工业界从事研究、生物技术和医学方面的职业。该项目还将扩大在该地区研究中心参与研究体验的本科生人数，并扩大公立学校K-8学生的数学和科学机会。在大肠杆菌中，复制的完成似乎涉及在切割、切除和连接多余区域之前，分叉会聚区域的短暂过度复制。通过监测大肠杆菌中完成的DNA序列的拷贝数，该研究将识别出精确完成复制能力受损的突变体。利用质粒小染色体，结合二维和一维琼脂糖凝胶分析将用于鉴定野生型和突变型细胞在完成复制过程中产生的结构中间体。最后，研究将确定完成机制是否在真核生物中是保守的，通过检查酵母中的同源突变体来确定是否在真核生物染色体上观察到复制分叉聚集的类似异常。

The mechanism by which chromosome replication is accurately completed is not well understood. To maintain genomic integrity during replication, cells must have an ability to recognize replicated regions, limit the ongoing replication process, and join converging daughter strands at the precise point at which the DNA sequence has doubled. Despite these complexities, this process occurs with remarkable efficiency (occurring thousands of times per division in human cells) and is fundamental to the accurate duplication of the genome. It appears to be a common point of deregulation in many cancer cells and is deliberately targeted for inactivation by viruses to induce the lytic phase of replication (wherein the virus multiplies rapidly). This project will determine how cells sense replicated regions and accurately complete the proess at the precise doubling point in the model organisms, Escherichia coli and Saccharomyces cerevisiae. Students trained during this project will be qualified for careers in research, biotechnology and medicine in both academia and industry. The project will also expand the number of undergraduates who participate in research experiences at research centers in the region and expand the math and science opportunities for K-8 students in the public school system.Completion of replication in E. coli appears to involve a transient over-replication of the region where forks converge before the excess regions are incised, resected, and joined. By monitoring the copy number of DNA sequences where completion occurs in E. coli, the research will identify mutants that have an impaired ability to accurately complete replication. Using plasmid minichromosomes, a combination of 2D and 1D agarose gel analysis will then be used to identify the structural intermediates that arise during the completion of replication in both wild type and mutant cells. Finally, the research will determine if the completion mechanism is conserved in eukaryotes, by examining the homologous mutants in yeast to identify whether similar abnormalities on eukaryotic chromosomes are observed where replication forks converge.