Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells.

Novel in vivo excision assays for monitoring the excised oligonucleotide products of nucleotide excision repair in UV-irradiated cells have provided unprecedented views of the kinetics and genomic distribution of repair events. However, an unresolved issue is the fate of the excised oligonucleotide products of repair and their mechanism of degradation. Based on our observation that decreases in excised oligonucleotide abundance coincide with the induction of apoptotic signaling in UV-irradiated cells, we considered the possibility that caspase-mediated apoptotic signaling contributes to excised oligonucleotide degradation or to a general inhibition of the excision repair system. However, genetic and pharmacological approaches to inhibit apoptotic signaling demonstrated that caspase-mediated apoptotic signaling does not affect excision repair or excised oligonucleotide stability. Nonetheless, our assay for detecting soluble DNAs produced by repair also revealed the production of larger DNAs following DNA damage induction that was dependent on caspase activation. We therefore further exploited the versatility of this assay by showing that soluble DNAs produced by both nucleotide excision repair and apoptotic signaling can be monitored simultaneously with a diverse set of DNA damaging agents. Thus, our in vivo excision repair assay provides a sensitive measure of both repair kinetics and apoptotic signaling in genotoxin-treated cells.

Preparation of soluble isotopically labeled human growth hormone produced in Escherichia coli.

Isotopically labeled proteins have been used as internal standards for mass spectrometry (MS)-based absolute protein quantification. Although this approach can provide highly accurate analyses of proteins of interest within a complex mixture, one of the major limitations of this method is the difficulty in preparing uniformly labeled standards. Human growth hormone (hGH) is one of the most important hormones that circulate throughout the body, and its measurement is primarily of interest in the diagnosis and treatment of growth disorders. In order to provide a useful internal standard for MS-based hGH measurement, we describe an efficient strategy to produce a potentially valuable, stable isotope-labeled hGH with high purity and yield. The strategy involves the following steps: solubilization of hGH under labeling conditions, detection of stable isotope incorporation, large-scale purification, analysis of the labeled protein, and assessment of the labeling efficiency. We show that the yield of soluble hGH under selective isotopic labeling conditions can be greatly increased by optimizing protein expression and extraction. Our efficient method for generating isotopically labeled hGH does not influence the structural integrity of hGH. Finally, we assessed the efficiency of stable isotope labeling at the intact protein level, and the result was further verified by amino acid analysis. These results clearly indicate that our labeling approach allows an almost complete incorporation of 13C615N4-arginine into the hGH expressed in E.coli without detectable isotope scrambling.