Functional impacts of the ubiquitin-proteasome system on DNA damage recognition in global genome nucleotide excision repair.

The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood. Here, we show that the proteasome subunit PSMD14 and the UPS shuttle factor RAD23B can be recruited to sites with UV-induced photolesions even in the absence of XPC, suggesting that proteolysis occurs at DNA damage sites. Unexpectedly, sustained inhibition of proteasome activity results in aggregation of PSMD14 (presumably with other proteasome components) at the periphery of nucleoli, by which DDB2 is immobilized and sequestered from its lesion recognition functions. Although depletion of PSMD14 alleviates such DDB2 immobilization induced by proteasome inhibitors, recruitment of DDB2 to DNA damage sites is then severely compromised in the absence of PSMD14. Because all of these proteasome dysfunctions selectively impair removal of cyclobutane pyrimidine dimers, but not (6-4) photoproducts, our results indicate that the functional integrity of the proteasome is essential for the DDB2-mediated lesion recognition sub-pathway, but not for GG-NER initiated through direct lesion recognition by XPC.

Hsp104 binds to yeast Sup35 prion fiber but needs other factor(s) to sever it.

The interaction of Hsp104 with yeast prion fibers made of Sup35NM, a prion-inducing domain of Sup35, was tested. When fluorescently labeled Hsp104 was added to the preformed fibers, individual fibers were fluorescently decorated uniformly along the fiber length. However, the density of fluorescence differed from one fiber to another, indicating the presence of subspecies of Sup35NM fibers. The time course of fiber formation from monomer Sup35NM was delayed by Hsp104. Hsp104-mediated fragmentation of fibers was tested using bead-tethered fibers. In contrast with the recent report (Shorter, J., and Lindquist, S. (2004) Science 304, 1793-1797), Hsp104 alone was unable to sever the fibers. Yeast cell lysate or the Hsp104-deficient cell lysate plus Hsp104 caused ATP-dependent, guanidine hydrochloride-sensitive fragmentation of the fibers. Thus, in our experimental setup, Hsp104 plus other factor(s) in the yeast cytosol are required for severing yeast prion fiber. The reason of discrepancy from the above report is unknown but is possibly caused by different conformational subspecies of prion fibers.

beta-Helix is a likely core structure of yeast prion Sup35 amyloid fibers.

We have studied the core structure of amyloid fibers of yeast prion protein Sup35. We developed procedures to prepare straight fibers of relatively uniform diameters from three kinds of fragments; N (1-123), NMp (1-189), and NM (1-253). X-ray fiber diffraction patterns from dried oriented fibers gave common reflections in all three cases; a sharp meridional reflection at 4.7A, and a diffuse equatorial peak at around 9A, apparently supporting the typical "cross-beta" structure with stacked beta-sheets proposed for many different amyloid fibers. However, X-ray fiber diffraction from hydrated fibers showed the meridional reflection at 4.7A but no equatorial reflections at 9A in all three cases, indicating that the stack of beta-sheets in dried fibers is an artifact produced by drying process. Thus, the core structure of these amyloid fibers made of the N domain is likely to be beta-helix nanotube as proposed by Perutz et al.