Single molecule analysis of Human DNA replication

All cells contain a complete copy of the organism's DNA, the genetic blueprint of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. This requires the process of DNA replication to start at thousands of sites across the chromosomes - called DNA replication initiation sites. Our project aims to determine the location of replication initiation sites in human cells. This is important because the location and distribution of replication initiation sites have been implicated in causing human diseases such as cancer.
It has been challenging to identify DNA replication initiation sites in human cells, because there is a lot of variability between cells. To date, most experiments have used the average from millions of cells, but this hides the variability between cells. We have developed a novel technology that identifies DNA replication initiation sites on thousands of single molecules that each originated in a single cell. Our approach can also identify specific sequences that are challenging to copy. This will allow us to test the hypothesis that some sites, thought to be involved in replication initiation, in fact are sites that impede DNA replication. Distinguishing between sites that initiate versus impede DNA replication will be crucial in understanding the causes of genetic instability that underlie some human diseases.

Complete, accurate genome replication is essential for life. Our long-term goal is to determine how cells faithfully complete genome replication. Complete genome replication is achieved by initiating DNA replication at multiple discrete sites. Both the characteristics of replication initiation sites and subsequent interruptions to replication have been implicated in human genetic and phenotypic variation. The location and distribution of replication initiation sites have been proposed to underlie genome instability and the biogenesis of tumorigenic translocations. However, widely used methods for detecting replication initiation sites in mammalian cells produce inconsistent results. Therefore, there is no consensus of where in the human genome DNA replication initiates. To tackle this problem, we have developed a single molecule sequencing technology that identifies replication initiation sites. Our preliminary data indicates that ~80% of initiation sites across the human genome are dispersed with only ~20% within zones that could be detected by existing population-level genomic approaches. In this project, we will comprehensively identify the location and nature of replication initiation sites in the human genome. In addition, we will test the hypothesis that some GC-rich sequences reported to direct replication initiation in fact are attributable to fork arrest and replication restart. Distinguishing between replication initiation and replication restart is crucial since such sites have been implicated in the biogenesis of tumorigenic translocations.