Evidence is accumulating to suggest that the response of mammalian cells to DNA damage may fall into two categories: responses that are activated by the presence of DNA damage itself and those that are activated as part of a more global stress-induced response. However, little progress has been made in understanding what the signal is that initiates some of these responses and what biological role the responses play. The long term objective of this proposal is to better understand the role that radiation modulated proteins (RMPs) play in human breast cells when these proteins are expressed at specific stages in the cell cycle. The proposed studies emerge from our preliminary findings that a novel excision repair process is induced in human cells when they are irradiated with gamma-rays at the G1/S border or in early S phase. These are the stages in the cell cycle in which cells are usually found to be radiation sensitive. The induced repair is distinguishable from constitutive excision repair in that a much longer repair patch is synthesized (150 vs 3 nucleotides). Our hypothesis is that these very long patches result from the repair of DNA lesions that are either unique to ionizing radiation or produced in a DNA structure specific to these stages in the cell cycle. The research described in this grant application will use both human mammary epithelial cells and human skin fibroblasts to study this induced repair process. The inclusion of human breast cells in our project will provide additional insights into the processing of DNA damage in these cells, and ultimately, a better understanding of breast cancer. This proposal is designed to: (l) Isolate and characterize the proteins induced in human mammary epithelial cells by ionizing radiation. Our primary strategy will be to identify the proteins specifically induced when cells at the G1/S border are exposed to gamma- rays and isolate the genes encoding them. (2) Determine the signal that leads to the production of very long excision repair patches. We will ascertain whether a specific class of radiation-induced lesion leads to the production of these long patches, or whether they result from the introduction of damage into DNA structures that are formed predominately at the onset of S phase, such as in transcriptionally """"""""bubbles"""""""" or in replication forks. (3) Examine the relationship between the induced long patch repair and constitutive excision repair pathways. We will examine whether the process that leads to very long repair patches plays a role in the repair of other radiation-induced lesions, such as thymine glycols. We will also determine whether the production of the very long repair patches is influenced by the repair competency of the cell by examining the phenomenon in xeroderma pigmentosum cells, which are defective in the nucleotide excision repair pathway.
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