ELA1 and CUL3 are required along with ELC1 for RNA polymerase II polyubiquitylation and degradation in DNA-damaged yeast cells.

Nucleotide excision repair (NER) is a versatile DNA repair process which operates on bulky helix-distorting lesions caused by a variety of DNA-damaging agents, including UV and chemical agents. In eukaryotes, following the initial recognition of the DNA lesion by the damage recognition factors, the damaged strand is unwound from the undamaged strand by the DNA helicases present in TFIIH to promote the dual incision of the damaged strand on the 5′ side and the 3′ side of the lesion by two separate endonucleases, which then results in the removal of a fragment of ∼30 nucleotides containing the lesion (21, 25). In eukaryotes, the genetic controls of NER for the nontranscribed regions and the transcribed regions of the genome differ in some important respects. For example, in the yeast Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for the repair of nontranscribed regions of the genome as well as for the repair of the nontranscribed DNA strand (21). The Rad7 and Rad16 proteins form a heterodimeric complex in which Rad16, a member of the Swi2/Snf2 family of ATPases, provides a DNA-dependent ATPase activity, and the Rad7-Rad16 complex binds preferentially to UV-damaged DNA in an ATP-dependent manner (8, 9). We previously suggested a role for the Rad7-Rad16 complex in the scanning of nontranscribed regions of the genome for DNA lesions and in initiating the nucleation of NER protein factors at the lesion site (8, 9). Rad16 also harbors a C3HC4 motif, a characteristic feature of ubiquitin (Ub) ligases; however, how Rad16 functions in such a role is not understood. DNA lesions from the transcribed strand of expressed genes are removed faster than lesions from the nontranscribed strand, a phenomenon known as transcription-coupled repair (TCR) (17, 18, 29). TCR is conserved from Escherichia coli to humans, and the process in E. coli, in which a transcription repair coupling factor promotes the release of the stalled RNA polymerase along with the nascent transcript from the damage site and then recruits the NER proteins for lesion removal, is well understood (27). In humans, TCR is modulated by Cockayne syndrome A and B proteins (CSA and CSB), and in yeast, TCR is modulated by Rad26, which is the CSB counterpart (21, 25). Although it is generally accepted that the stalled RNA polymerase II (Pol II) has to be removed from the lesion site for the repair machinery to assemble there, it is not known whether Pol II is removed or translocated away from the lesion site or whether it undergoes some sort of conformational change to allow the assembly of NER proteins at the lesion site. Another phenomenon that is observed upon treatment of yeast or human cells with DNA-damaging agents is lysine 48-linked polyubiquitylation of the Rpb1 subunit of Pol II, which targets Pol II for proteasomal degradation (6, 23, 24). It remains unclear, however, whether Pol II polyubiquitylation contributes to TCR by targeting it for degradation, thereby promoting the assembly of the NER proteins at the lesion site. Moreover, since the Ub ligase (E3) responsible for Pol II polyubiquitylation in yeast or human cells has not been identified, this phenomenon has not been amenable to genetic analysis. Our recent observation that elongin C (Elc1) was required for Pol II polyubiquitylation and degradation in yeast cells raised the possibility that an Elc1-dependent Ub ligase might be involved in this process (24). Yeast Elc1 is a homolog of mammalian elongin C (1), which forms a heterotrimeric complex with elongins A and B (3). In mammalian cells, the elongin A, B, and C complex increases the rate of transcription elongation by suppressing Pol II pausing (4, 5). In yeast cells, however, the only elongins present are elongins A and C, and the complex of these two proteins does not stimulate transcription elongation by Pol II (14). Since Elc1 is required for Pol II polyubiquitylation and degradation in yeast cells (24), and since it exists in vivo in a complex with the Rad7 and Rad16 proteins (22), we first considered the possibility that this protein complex was involved in modulating Pol II modification. The Elc1-Rad7-Rad16 complex shares several characteristic features of the SCF (Skp1-Cullin-F box)-type Ub ligases (15). In the SCF complex, the Skp1 subunit acts as an adaptor which connects the F-box protein to the scaffold protein Cdc53/cullin, to which the RING-H2 finger protein Rbx1/Roc1 binds. The F-box protein binds the protein substrates targeted for ubiquitylation by the Ub-conjugating enzyme (E2) which assembles with the Cdc53/cullin subunit. In the Rad7-Rad16-Elc1 complex, Rad7 is an F-box protein, Rad16 is a RING-H2 protein, and Elc1 is a Skp1 homolog. However, our finding that Pol II polyubiquitylation and degradation were not affected in rad7Δ or rad16Δ yeast cells treated with UV or 4-NQO (4-nitroquinoline-1-oxide) (24) led us to consider other possibilities. Here we show that elongin A (Ela1) and cullin 3 (Cul3) are required for Pol II polyubiquitylation and degradation in DNA-damaged yeast cells, and based upon these and other observations, we propose that an E3 comprised of Elc1, Ela1, Cul3, and Roc1 (Rbx1) mediates this process in yeast cells. Further, our genetic analyses indicate that Pol II polyubiquitylation and degradation are not a prerequisite for Rad26-mediated TCR; rather, these two processes provide separate means of lesion removal from the transcribed strand. In addition, these observations lead us to predict that in human cells, the von Hippel-Lindau (VHL) tumor suppressor complex (28, 32), comprised of elongin B, elongin C, cullin 2, and Rbx1 subunits, would function in an analogous manner in mediating Pol II polyubiquitylation and degradation.