No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

APLF is a forkhead associated (FHA) domain-containing protein with unique poly(ADP)-ribose (PAR)-binding zinc finger (PBZ) domains that are involved in the DNA damage response. The interaction of the APLF PBZ domains with PAR is essential for the rapid recruitment of APLF to sites of DNA double strand breaks (DSBs), while the FHA domain facilitates non-homologous end-joining. In response to ionizing radiation (IR), APLF is phosphorylated at Serine-116 (APLFS116), although the function of this post-translational modification has yet to be defined. Here we provide a detailed characterization of the IR-induced and ATM- dependent phosphorylation of endogenous APLF at Serine-116 in the context of DNA damage. We additionally examine a novel APLF FHA-dependent interaction with 53BP1 (p53 Binding protein 1). Together, we illustrate that APLFS116 phosphorylation is dependent upon both the tandem PBZ domains, as well as the FHA-domain, and that the depletion of either PARP3 or 53BP1, similarly affected APLFS116 phosphorylation. Furthermore, we show that DSB-repair was compromised in cells expressing the APLFS116A mutation. Collectively, our findings provide a detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that APLFS116 phosphorylation facilitates APLF-dependent DSB repair.

APLF is a forkhead associated (FHA) domain-containing protein with unique poly(ADP)-ribose (PAR)-binding zinc finger (PBZ) domains that are involved in the DNA damage response. The interaction of the APLF PBZ domains with PAR is essential for the rapid recruitment of APLF to sites of DNA double strand breaks (DSBs), while the FHA domain facilitates non-homologous end-joining. In response to ionizing radiation (IR), APLF is phosphorylated at Serine-116 (APLFS116), although the function of this post-translational modification has yet to be defined. Here we provide a detailed characterization of the IR-induced and ATM- dependent phosphorylation of endogenous APLF at Serine-116 in the context of DNA damage. We additionally examine a novel APLF FHA-dependent interaction with 53BP1 (p53 Binding protein 1). Together, we illustrate that APLFS116 phosphorylation is dependent upon both the tandem PBZ domains, as well as the FHA-domain, and that the depletion of either PARP3 or 53BP1, similarly affected APLFS116 phosphorylation. Furthermore, we show that DSB-repair was compromised in cells expressing the APLFS116A mutation. Collectively, our findings provide a detailed understanding of the molecular pathway that leads to the phosphorylation of APLF following DNA damage and suggest that APLFS116 phosphorylation facilitates APLF-dependent DSB repair.

The reaction of polycyclic hydrocarbon epoxides with the phosphate group of DNA has been suggested as potentially important in the cancer process initiated by this class of compounds. Herein the chemical reactions of model phosphates with various classes of epoxides are investigated. With aliphatic and non-bay-region type expoxides the reaction occurred by stereospecific trans addition of phosphate on the epoxide. Various physical-chemical aspects of this reaction are investigated. With K-region type epoxides the same reaction gave exclusively phenols. The biological implications of this result are discussed. Finally, bay-region type oxides, on reaction with phosphate, gave phosphotriesters whose stabilities depend on the phosphodiester nucleophile used and on the presence and arrangement of the diol grouping next to the epoxide.

Understanding the binding specificity of transcription factors is an important step towards accurate computational prediction of regulatory sequences governing gene expression. Higher-throughput binding site characterization methods have long been available in the laboratory for the study of protein-DNA interactions in solution or upon a surface. In this thesis a new method is introduced for characterization of inducible regulatory sequences in living cells based on construction and analysis of promoter-reporter gene plasmids. Spiked oligonucleotides are used to generate libraries of regulatory sequences with subtle variations from a known regulatory element. Screening of the library in cell culture for the capacity of the mutated sequences to mediate expression provides a diverse collection of responsive and non-responsive sequences to aid in understanding the sequence requirement for an inducible transcription factor binding site. We apply the methodology to the study of antioxidant responsive elements, the target sites of the Nfe212 transcription factor. These target sequences commonly found in the promoters of detoxification genes modulate gene expression in response to a diverse array of chemicals. The variants serve as a primary screen for future targeted mutational analysis to further characterize context-specific sequence requirement in the ARE and/or interdependence between positions. Moreover, a transcription factor binding profile can be generated from functional ARE variants in the library screen. Such an ARE profile performs as well as standard profiles based on bona fide ARE sequences drawn from the scientific literature.