This protocol requires labeling DNA damage with the addition of fluorescently tagged nucleotides to free DNA ends

A distinct group of these disorders are prion diseases caused by an infectious amyloid form of the prion protein, called PrPSc. Self-perpetuating prion amyloids have also been described in lower eukaryotes, yeast Saccharomyces cerevisiae - and filamentous fungus Podospora anserina, in which they determine nonchromosomally inherited phenotypes. Notably, in spite of significant progress in discovering amyloids, most of them were found by analyzing PB 203580 proteins with sequences similar to known amyloid-forming proteins, or via timeconsuming and sometimes highly sophisticated genetic screenings for factors that determine non-Mendelian traits,,,. This suggests that development of a reliable and universal biochemical approach for fast identification of novel amyloid-forming proteins is both important and timely. To date, biochemical approaches used to identify novel amyloid proteins have been based on their common ability to form insoluble aggregates. For example, the prion form of PrP was detected as a major protease-resistant protein component of infectious, high molecular weight aggregates isolated by ultracentrifugation. Amyloid beta peptide was extracted from a protein pellet fraction obtained from brain homogenate of a patient, who had died of Alzheimer’s disease. Unfortunately, such approaches can only be used for identification of amyloids that form large deposits. A common property of amyloid aggregates is resistance to various detergents. Amyloid nature of such detergent-insoluble aggregates is supported by the fact that they often contain generic amyloid epitopes for DNA aptamer binding. High resistance of amyloids to treatment with detergents allows amyloid isolation from yeast cell lysates by sedimentation in the presence of SDS. A validation of this method carried out for the yeast prion, demonstrated its suitability for identification of proteins forming relatively abundant amyloids. An alternative approach based on separation of SDS-insoluble amyloids by their inability to enter polyacrylamide gel and subsequent mass-spectrometry allowed the identification of the Rnq1 and Ure2 prion proteins, which are less abundant than Sup35. Nevertheless, both these methods enable only identification of proteins comprising amyloids that are resistant to treatment with SDS, while some amyloids, which are not enriched in Q or N residues, such as amyloids of Ab, are soluble in the presence of SDS. Here, we present a novel proteomic approach for screening for amyloid proteins, called PSIA. This approach can use either SDS or sarkosyl for amyloid isolation. We show that the use of sarkosyl instead of SDS for the purification of amyloid aggregates allows isolation of amyloids which cannot withstand SDS treatment, thus making the approach more universal than those which were developed earlier. This approach was validated in a yeast model by detection of prion proteins and mammalian amyloid-forming proteins. We also identified several yeast proteins which form detergent-insoluble aggregates in response to expression of human huntingtin with an expanded polyglutamine domain. The PSIA approach developed in this work allowed us to detect all the tested proteins with well established capability for amyloid formation, such as yeast Rnq1 and Sup35 prion proteins as well as mammalian PrP, Ab and mutant huntingtin tagged with GFP. Notably, the obtained results depended on the detergent used for amyloid isolation. The use of either SDS or sarkosyl allowed detection of Rnq1, Sup35 and Q103-GFP, while PrP-GFP and Ab-GFP were detected only with the use of sarkosyl, because their aggregates are not resistant to SDS treatment. Solubility in 1% SDS was earlier shown for Ab aggregates formed in mammals. Thus, the use of sarkosyl for aggregate isolation enables identification of amyloid proteins which cannot be isolated with the use of SDS. It is also worth to stress that our results provide new insight into resistance of amyloids to detergents and solvents. The only characteristic shared by all tested amyloids, is the resistance to treatment with 3% sarkosyl at room temperature. Also, some amyloids, such as prion polymers of Sup35, are insoluble in UTC buffer used for 2D-DIGE, which contains chaotropic agents, and even in formic acid, but can be efficiently dissolved by boiling in the presence of 2% SDS. This complicates the method, since solubilization of various amyloids will requires different solvents. Another limitation of PSIA is that extremely acidic and basic proteins cannot be detected by 2D-DIGE.

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