Transcription-coupled repair in yeast is independent from ubiquitylation of RNA pol II: Implications for Cockayne's syndrome

Abstract

Cockayne's syndrome cells lack transcription-coupled nucleotide excision repair (TCR) and ubiquitylation of RNA polymerase II large subunit (RNA pol II LS), suggesting that ubiquitylation of RNA pol II LS may be necessary for TCR in eukaryotes. Rsp5 is the sole yeast ubiquitin-protein ligase that ubiquitylates RNA pol II LS in cells exposed to DNA-damaging agents. In yeast lacking functional Rsp5, there is no ubiquitylation of RNA pol II LS. We show here that removal, repression, or over-expression of Rsp5 has no effect on TCR, demonstrating that ubiquitylation of the RNA pol II LS is not required for TCR. We infer that the lack of ubiquitylation of RNA pol II LS in Cockayne's syndrome cells does not cause their defect in TCR. C ockayne's syndrome (CS) is a rare autosomal recessive disorder displaying a variety of symptoms, including severe neurological abnormalities, dwarfism, deficiency of subcutaneous fat, and sun sensitivity (1). The UV sensitivity of CS patients and their cells in culture is thought to result from a deficiency in RNA synthesis (2), likely because of an inability to preferentially remove transcription-blocking DNA damage from the transcribed strands of expressed genes: i.e., impaired transcriptioncoupled repair (TCR) (3, 4).It has been shown that HeLa cells and normal human fibroblasts in culture ubiquitylate the large subunit of RNA polymerase II (RNA pol II LS) after exposure to UV radiation, cisplatin, mitomycin C, and methyl methanesulfonate (5). The ubiquitylation of RNA pol II LS was absent in UV-irradiated fibroblasts from CS patients. Interestingly, fibroblasts from patients displaying another autosomal recessive disorder associated with a DNA repair deficiency, xeroderma pigmentosum, were capable of ubiquitylating RNA pol II LS after UV irradiation (6). In both normal and xeroderma pigmentosum fibroblasts, the ubiquitylated form of RNA pol II LS was hyperphosphorylated, a form that is associated with the elongating transcription complex.Ubiquitin is a 76-amino acid peptide that gets linked via its terminal glycine (residue 76) to a cysteine residue of the ubiquitin-activating protein (E1) via a thioester bond. The ubiquityl moiety is then moved via transesterification from the E1 enzyme to a cysteine residue of one of the ubiquitin-conjugating enzymes (E2). Ubiquitin is then transferred via further transesterification to one or more -lysine residues in the acceptor or target protein. This step may require a ubiquitin-protein ligase, or E3. Finally, the recently identified E4 proteins bind ubiquitylated proteins and, together with E1, E2, and E3, facilitate formation of multiubiquitin chains on substrate proteins (7,8). Substrates of the ubiquitin system are degraded by the large 26S proteasome in an ATP-dependent fashion. Ubiquitylation may also serve a regulatory function independent of proteolysis. Ubiquitylation, through proteolysis and other mechanisms, plays a regulatory role in the cell cycle, cellular differentiation, stress responses, and many other cellular processes.In the...

Schizosaccharomyces pombe Ddb1 Recruits Substrate-Specific Adaptor Proteins through a Novel Protein Motif, the DDB-Box

“…Second, a DDB1 sequence homolog has not been identified. Finally, Rsp5, the only E3 which ubiquitinates Rpb1 in S. cerevisiae, is also dispensable for TCR (31). In the course of UV survival assays, we noticed that the UV survival of the ⌬ddb1 ⌬uvde and ⌬rhp26 ⌬uvde mutants were not similar (Fig.…”

Rescue of arrested RNA polymerase II complexes

“…Important in this connection, Pol II becomes ubiquitylated and degraded in response to DNA damage (Beaudenon et al, 1999;Bregman et al, 1996;Luo et al, 2001;Ratner et al, 1998), which raises the possibility that it is removed from lesion sites so that rapid repair can take place. However, enabling TCR is unlikely to be the function of Pol II proteolysis (Lommel et al, 2000;Woudstra et al, 2002). Rather, a new study suggests that ubiquitylation and Pol II degradation in response to DNA damage constitutes an alternative to DNA repair -a last resort (Woudstra et al, 2002).…”

Influence of irofulven, a transcription-coupled repair-specific antitumor agent, on RNA polymerase activity, stability and dynamics in living mammalian cells

“…More recent data indicate that degradation of the Pol II LS is not an absolute requisite for repair of cisplatin adducts, at least in model systems using cellular extracts from human cells (Tremeau-Bravard et al, 2004;Sarker et al, 2005;Lainé and Egly, 2006a). Furthermore, yeast mutants lacking ubiquitin ligase, and thus unable to ubiquitylate the stalled Pol II LS, are still TCR proficient (Lommel et al, 2000). Furthermore, it has been shown that proteolysis of the Pol II LS can occur independently of the TCR process as treatment with alpha-amanitin or nucleotide starvation is accompanied by ubiquitylation and proteolysis of the polymerase (Lee et al, 2002;Yang et al, 2003).…”