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A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately undergraduate institution, and to complete an entire research project in a realistic timeframe via a time-intensive curriculum as a special summer session. The course content gives a strong background in protein structure/chemistry, purification principles, protocol development, optimization strategies, use and programming of an automated chromatography instrument, and characterization strategies with an emphasis on X-ray crystallography. The laboratory portion offers students the chance to purify a protein (human inosine triphosphate pyrophosphatase) from start to finish, program and use an Ä KTA fast protein liquid chromatography instrument, and to grow and analyze their own protein crystals using their purified protein. This innovative laboratory experience gives the participating students the opportunity to complete a miniresearch project in real time and enhances their overall understanding of important biochemical research techniques and the instrumentation involved, fostering a better understanding of the research process all within a classroom setting. Evaluations and feedback concerning this course indicated a positive learning environment, a retention of knowledge and skills, a belief that the skill set learned continues to be useful in current endeavors, and a sense of accomplishment in the completion of an actual research project within the confines of a class setting.Keywords: Active learning, curriculum design development and implementation, laboratory exercise, new course development, protein structure function and folding.One major downfall in any undergraduate laboratory course is the disconnection between a series of 3-hour miniaturized experiments and the realistic nature of true bench work. In a typical undergraduate course, the experiments are written to introduce students to working at a bench, calculations, and use of equipment, but are all designed with maximal successful outcome ratios for the students. In an actual research laboratory setting, students must deal with experiments that take extended timeframes to complete, multiple trials for optimization, and work in collaboration to optimize equipment and reagent use. Students quickly learn that research results are not as easily obtained as their former laboratory manuals made it seem. Many of our science majors at the undergraduate level are hoping to join graduate programs or work forces which require applicants with research experience, who are detailed notebook writers, and who have developed solid critical thinking skills. Graduate students may find that their research project involves the need for protein isolation and/or purification, a skill set that may be outside of their laboratory's area of...

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-stranded break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phase of the cell cycle and all three subunits of RPA contain phosphorylation sites. The exact set of HR-relevant phosphorylation sites of RPA is unknown. In this study, the phosphorylation sites of chromatin-bound RPA in S and G2 phase were identified. After DNA damage, phosphorylation included pSer4/8, pSer12, pThr21, pSer23 and pSer33 of RPA2. Phosphorylation of these sites increased with time after DNA damage. Using a general ATM/ATR phosphorylation antibody, only RPA2 had substantial pSer/pThr ATM/ATR signals. Additionally, pTyr was detected on RPA1 and RPA2 and was removed in response to DSBs, indicating the regulatory action of a phosphatase. A capillary isoelectric focusing immunoassay used under native conditions revealed in high resolution isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Strikingly, RPA is a phosphoprotein in control G2 cell lysates with up to 5 putative phospho-isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33 and RPA1 pTyr. DNA damaged lysates contained 9 isoforms, containing up to 14 phosphates. DNA damage isoforms contained RPA2 pSer4/8, pSer12, pThr21, pSer23, pSer33 and other not yet identified phosphorylation sites. Citation Format: Kerry Brader, Adam Mosel, Shengqin Liu, Elizabeth Kremmer, Kaitlin Goettsch, Heinz-Peter Nasheuer, Greg Oakley, Gloria Borgstahl. Interplay of DNA damage and cell cycle signaling on Replication Protein A in human cells. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 33. doi:10.1158/1538-7445.CANSUSC14-33