Cancer Susceptibility and S Phase Initiation Sites

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DESCRIPTION (provided by applicant): Deletions and sequence mutations of genes are the mechanisms typically considered when evaluating the etiology and progression of neoplasia. This topic has been extensively examined over the past several decades. Likewise, genetic instability as manifested by development of numerical and structural alterations of chromosomes is also a typical feature of cancer development that has been the subject of intense study. More recently it has been recognized that many cancers manifest alterations in gene expression that are not coincident with gene mutations. These changes are thought to be associated with alterations of DNA methylation in the promoter of these genes and by histone modifications at the sites of these promoters. There have been far fewer efforts to identify the mechanisms that can explain these latter types of cancer-related changes. In this project we seek to understand mechanisms that can explain the transition of alleles of non-imprinted genes from synchronous to non-synchronous replication timing in carcinogenesis. While it is known that such shifts in replication timing can lead to the alterations in gene expression seen in many types of cancers, the exact mechanisms responsible for the conservation of replication timing are not known. Others and we found that both DNA replication and transcription are often initiated in or near the gene promoter and associated CpG island. In this project we seek to better define the essential genomic elements required for the function of DNA replication origins found at transcriptional promoters. We also pose the following questions: Do differences in origin firing times correspond to differences in the binding time of specific factors necessary for origin activation? Are there sites in normal human cells that slow or pause the progression of DNA replication forks? Are such sites involved in the regulation of the order of activation time of adjacent origins of DNA replication? To gain insights into these mechanisms and answer these questions we propose the following Specific Aims. Specific Aim 1: Characterize genetic elements necessary for activity of the HPRT origin of replication. Specific Aim 2: Analyze binding of the pre-replication complex at the HPRT and G6PD origins of replication on the active and the inactive X chromosome in female human cells. Specific Aim 3: Analyze the architecture of replication and potential fork progression barriers in an early replicated region of the human genome.