Quantitative measurement of translesion DNA synthesis in mammalian cells.

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism, in which specialized low-fidelity DNA polymerases bypass lesions that interfere with replication. This process is inherently mutagenic due to the miscoding nature of DNA lesions, but it prevents double strand breaks, genome instability, and cancer. We describe here a quantitative method for measuring TLS in mammalian cells, based on non-replicating plasmids that carry a defined and site-specific DNA lesion in a single-stranded DNA region opposite a gap. The assay is responsive to the cellular composition of TLS DNA polymerases, and TLS regulators. It can be used with a broad variety of cultured mammalian cells, and is amenable to RNAi gene silencing, making it a useful tool in the study of TLS in mammalian cells.

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity.

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.

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.

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 Pcna(K164R/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 Pcna(K164R/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.

Mechanism of rapid transcriptional induction of tumor necrosis factor alpha-responsive genes by NF-kappaB.

NF-kappaB induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-kappaB-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-kappaB interacts directly with TAF(II)s, induction of NF-kappaB by tumor necrosis factor alpha (TNF-alpha) does not enhance TFIID recruitment and preinitiation complex formation on some NF-kappaB-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-alpha stimulus. Using the immediate-early TNF-alpha-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-kappaB. In vitro transcription assays confirmed that NF-kappaB 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-kappaB on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.

AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation.

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.

Marker-fusion PCR for one-step mutagenesis of essential genes in yeast.

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.