CANCER SUSCEPTIBILITY AND S PHASE INITIATION SITES

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Mammalian cells are most vulnerable to carcinogenesis when exposed to DNA damaging agents at the very beginning of the S phase during synchronized proliferation. The applicants hypothesize that this vulnerability may be associated with carcinogen-induced alterations of regions of DNA that regulate the origins of replication that control the start of the S phase. Thus applicants seek to find origins of replication that are activated in early S phase. Despite recent advances in understanding how DNA replication is initiated in prokaryotes, viruses and yeast, little is known about how DNA replication is initiated at the start of S phase and how multiple subsequent origins are activated in a regulated, sequential manner in mammalian cells. There is very limited knowledge of what constitutes an origin of replication in higher eukaryotes. The complex genome of human cells may utilize different types of replication origins in a coordinated and progressive process of replication during S phase. Origins that are activated as cells enter S phase might belong to a different class than those that activate later, resulting in orderly duplication of the genome. This project proposes a strategy to begin to address this issue. The applicants constructed a cosmid library of DNA sequences that are replicated as normal human fibroblasts (NHF1) enter S phase. They will search these libraries for clones with high potential of harboring origins of replication. Early replicating DNA and nuclear matrix-associated DNA will be used to identify clones that hybridize well with both types of probes (Aim 1). The ends of the cloned human DNA inserts will be sequenced and this information used to retrieve longer sequences deposited in DNA databases. The sequences will be analyzed for clustering of structural motifs that predict the potential for replication origin function (Aim 2). To confirm the potential of these sequences to serve as replication origins, a functional assay will be used based on competitive PCR to measure their increased relative abundance in small nascent DNA from proliferating human fibroblasts (Aim 3). Candidate clones also will be used to determine whether other cell types (lymphoblastoid and epithelial cells) share the same origins of replication at the start of S (Aim 4). Ultimately the applicants wish to determine whether changes in sequences at or near the initial origins of replication are a common feature of cancer cells. Such lesions could explain their observation of vulnerability to carcinogenesis in the early S phase. Mutations in regulatory sequences that control the initiation of replication at the start of the S phase may represent a mechanism by which the regulation of cell proliferation is relaxed. These alterations may be another type of genetic change that initiates the process of carcinogenesis.