Throughout their lives cells have to constantly monitor and repair the damage inflicted on their DNA. Free radicals and peroxides, generated during normal physiological processes and inflammation, as well as environmental pollutants, ultra-violet light, and ionizing radiation are the most common sources of DNA damaging agents. If not sensed and properly repaired, this damage can cause malignant transformation, in part through mutations at specific sites in certain oncogenes. In normal human cells, the genotoxic stress response is rather complex. It includes the induction of several genes that have been associated with a number of important cellular events such as cell cycle control, signal transduction, replication, mutagenesis, transcription, DNA repair and viral activation. We propose to study a particular aspect of the genotoxic stress response involving the induction of RNA-binding activity of specific proteins. Our initial data demonstrated for the first time that RNA-binding activity of cellular proteins can be inducible by DNA damage. This suggested important implications for the regulation of cellular genes after genotoxic stress, as well as possible involvement in DNA damage recognition and repair. The inducible binding activity of these proteins, as previously described constitutive RNA Binding Proteins (RBP), could regulate gene expression at the level of RNA processing and mRNA stability. More importantly, these RBP recognized a specific RNA structure, double stem loop. It is conceivable that such structures could be formed by nascent transcripts after partial separation from their DNA template. At sites of damage, transcription of these nascent transcripts might stall. By binding to these pre-mature sequences, the RBP may act as terminators and facilitate some aspect of DNA repair. Interestingly, the RNA structure recognized by these RBP is also reminiscent of the viral TAR RNA structure generated in HIV-1 infected cells. A role in the regulation of viral activation following genotoxic stress exposure seems also possible for these RBP. Using an original Northwestern blotting approach, we have detected a variety of RBP in whole nuclear extracts. A 40 kDa protein has been isolated by affinity chromatography using a specific RNA oligomer column. We plan to sequence this protein and isolate its cDNA in order to determine its role in the genotoxic stress response. We also plan to study the role of A-18, an independently isolated clone coding for a RBP induced by ultra-violet light, in the stress response.
