Endoplasmic reticulum-associated degradation (ERAD) disposes of aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated via the Sec61p translocon from the endoplasmic reticulum to the cytosol, where they are ubiquitinated and degraded by the proteasome. Since the Sec61p channel is also responsible for import of nascent proteins, this bidirectional passage should be coordinated, probably by molecular chaperones. Here we implicate the cytosolic chaperone AAA-ATPase p97/Cdc48p in ERAD. We show the association of mammalian p97 and its yeast homologue Cdc48p in complexes with two respective ERAD substrates, secretory immunoglobulin M in B lymphocytes and 6myc-Hmg2p in yeast. The membrane 6myc-Hmg2p as well as soluble lumenal CPY*, two short-lived ERAD substrates, are markedly stabilized in conditional cdc48 yeast mutants. The involvement of Cdc48p in dislocation is underscored by the accumulation of ERAD substrates in the endoplasmic reticulum when Cdc48p fails to function, as monitored by activation of the unfolded protein response. We propose that the role of p97/Cdc48p in ERAD, provided by its potential unfoldase activity and multiubiquitin binding capacity, is to act at the cytosolic face of the endoplasmic reticulum and to chaperone dislocation of ERAD substrates and present them to the proteasome.Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control process that selectively eliminates aberrant proteins in the secretory pathway. Protein substrates of ERAD are dislocated from the ER to the cytosol, where they are ubiquitinated and degraded by the proteasome (5). The Sec61p translocon is involved both in the import of nascent proteins into the ER and in dislocation of aberrant proteins from the ER. These two activities of Sec61p are mechanistically different because they involve distinct domains within Sec61p and dislocation-defective mutants of Sec61p are still proficient in protein import (40,50,56).Since nascent and aberrant proteins pass through the same Sec61p translocon, this bidirectional passage requires coordination. Moreover, the Sec61p translocon is a passive conduit; thus, the driving force to move polypeptides across it should be provided by accessory proteins. Indeed, passage through the Sec61p translocon requires molecular chaperones, and their contribution further illustrates that import and dislocation must be mechanistically distinct. For example, of the two hsp70s involved in import in yeast, BiP/Kar2p in the ER lumen and Ssa1p in the cytosol, mutants of BiP/Kar2p that are defective in dislocation are still proficient in import, and mutation in SSA1 does not affect degradation of the ERAD substrates pro-␣-factor and A1PiZ (9). Although the kar2 mutant initially used to link BiP to CPY* dislocation and degradation was also defective in protein import (41), the kar2 mutants that are defective only in dislocation of pro-␣-factor and A1PiZ directly demonstrate the role of Kar2p in dislocation (9). Furthermore, chaperones that are required for ERA...

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in PcnaK164R/K164R cells, which are resistant to PCNA ubiquitination, compared to Pcna+/+ cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in PcnaK164R/K164R mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.

NF-B induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-B-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-B interacts directly with TAF II s, induction of NF-B by tumor necrosis factor alpha (TNF-␣) does not enhance TFIID recruitment and preinitiation complex formation on some NF-B-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-␣ stimulus. Using the immediate-early TNF-␣-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-B. In vitro transcription assays confirmed that NF-B plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-B on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.The family of NF-B transcription factors is a central component of the cellular response to a broad range of extracellular signals, many of them are related to immunological functions and stress. NF-B controls the expression of a large number of genes including inflammatory cytokines, chemokines, immunological factors, adhesion molecules, cell cycle regulators, and pro-and antiapoptotic factors (24). A major pathway regulating NF-B activity involves its nuclear transport. In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form by IB proteins. Signals that activate NF-B trigger ubiquitination and degradation of IB by the proteosome, resulting in transport of NF-B into the nucleus and transcriptional activation of responsive genes. Since IB␣ is one of the NF-B target genes, the newly synthesized IB␣ negatively regulates NF-B, thus forming an autoregulatory loop.In the nucleus, transcriptional activation by NF-B involves its association with multiple coactivators. We reported previously that the substoichiometric TFIID subunit, TAF II 105, which is enriched in B cells, interacts directly with p65/Re1A, a member of the NF-B family, and is important for activation of a subset of NF-B-dependent antiapoptotic genes in vivo (30,36,37). Likewise, other TFIID subunits such as hTAF II 250, hTAF II 80, and hTAF II 28 were reported to bind to p65/Re1A (8), although the physiological importance of these interactions was not investigated. In addition to TFIID, the coactivator protein CREB-binding protein CBP and its homolog p300 were reported to be involved in transcription activation by the p65/Re1A subunit of NF-B (6, 25). p65 was also found to interact specifically with the composite coactivator ARC/DRIP, and this complex supports NF-B-dependent transcriptional activation in v...

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.

We describe a one-step gene replacement method based on fusion PCR that can be used to mutagenize essential genes at their endogenous locus. Marker-fusion PCR can facilitate transfer of alleles between strains as well as PCR-based techniques, such as site-directed and error-prone PCR mutagenesis, all without cloning or strain constructions. With this method, PCR is used to fuse a mutagenized fragment to an overlapping fragment containing a selectable marker flanked by regions of homology to the target. By transforming yeast with these PCR products, specific mutations are introduced at the endogenous locus through homologous recombination. We tested the 'marker-fusion PCR' method using the budding yeast CDC28 gene and were able to efficiently introduce site-directed mutations and integrate genomic or plasmid-borne mutant alleles. As a further application for this method, we used a spiked oligonucleotide to randomize the coding sequence for a single domain of CDC28 and were able to construct highly mutagenized libraries for this region.