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Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26
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DNA Repair xxx (2015) xxx–xxx
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Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26 Shisheng Li ∗ Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA
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Article history: Available online xxx Keywords: Rad26 RNA polymerase II Saccharomyces cerevisiae Transcription coupled nucleotide excision repair Transcription elongation factors
a b s t r a c t Transcription coupled nucleotide excision repair (TC-NER) is believed to be triggered by an RNA polymerase stalled at a lesion in the transcribed strand of actively transcribed genes. Rad26, a DNA-dependent ATPase in the family of SWI2/SNF2 chromatin remodeling proteins, plays an important role in TC-NER in Saccharomyces cerevisiae. However, Rad26 is not solely responsible for TC-NER and Rpb9, a nonessential subunit of RNA polymerase II (RNAP II), is largely responsible for Rad26-independent TC-NER. The Rad26dependent and Rpb9-dependent TC-NER have different efficiencies in genes with different transcription levels and in different regions of a gene. Rad26 becomes entirely or partially dispensable for TC-NER in the absence of Rpb4, another nonessential subunit of RNAP II, or a number of transcription elongation factors (Spt4, Spt5 and the RNAP II associated factor complex). Rad26 may not be a true transcription-repair coupling factor that recruits the repair machinery to the damaged sites where RNAP II stalls. Rather, Rad26 may facilitate TC-NER indirectly, by antagonizing the action of TC-NER repressors that normally promote transcription elongation. The underlying mechanism of how Rad26 functions in TC-NER remains to be elucidated. © 2015 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5.
6.
7. 8.
Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The involvement of Rad26 in TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Properties of Rad26 and its human homolog CSB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Other functions of Rad26 and CSB that may or may not be directly related to TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Recovery of RNA synthesis after UV irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.2. Transcription elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Chromatin remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Repressors of TC-NER in cerevisiae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 6.1. Rpb4/Rpb7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. Spt4/Spt5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.3. RNAP II associated factor 1 complex (Paf1C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Possible ways for Rad26 to facilitate TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Prologue
∗ Correspondence address. Fax: +1 225 578 9895. E-mail address: [email protected]
My postdoc experience at Michael (Mick) Smerdon laboratory greatly influenced my research career. Mick is very passionate about how DNA repair takes place in chromatin. However, he is not the “my way or the highway” kind of person, and allows and encourages lab people to explore other avenues of research related
http://dx.doi.org/10.1016/j.dnarep.2015.09.006 1568-7864/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: S. Li, Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.09.006
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to DNA repair. He frequently forgets my and other people’s names but pays great attentions to experimental and data details. He is a firm believer that “bad data is worse than no data at all”. 2. Introduction Nucleotide excision repair (NER) is a highly conserved multistep process that removes bulky and/or helix-distorting DNA lesions, such as UV induced cyclobutane pyrimidine dimers (CPDs) [1] (also see elsewhere in this issue). Transcription coupled NER (TC-NER) is the NER subpathway that is dedicated to rapid removal of lesions in the transcribed strand of actively transcribed genes. Global genomic NER (GG-NER), on the other hand, refers to NER throughout the genome. The two NER subpathways rely on different proteins for recognition of DNA lesions but share common factors in the later steps of the repair process. In Saccharomyces cerevisiae, Rad26 plays an important role in TC-NER, whereas Rad7, Rad16 and Elc1 are specifically required for GG-NER. In this minireview, I will summarize findings about how Rad26 may be implicated in TC-NER. 3. The involvement of Rad26 in TC-NER Rad26 was found to play an important role in TC-NER in S. cerevisiae [2]. However, disruption of RAD26 in otherwise wild type yeast cells does not cause detectable UV sensitivity. rad16 rad26 or rad7 rad26 cells are more UV sensitive than the single rad7 or rad16 cells but not as UV sensitive as cells that are entirely defective in NER (e.g., rad14) [3]. The mild effect of Rad26 on UV sensitivity may be due to the fact that Rad26 is not solely responsible for TC-NER and the very efficient GG-NER in yeast may largely compensate for the loss of Rad26-dependent TC-NER for cell survival [1]. The Rad26-independent TC-NER was found to be dependent on Rpb9, a nonessential subunit of the 12 subunit (Rpb112) RNAP II [4]. Indeed, like rad16 rad26 cells, rad16 rpb9 cells are moderately more UV sensitive than rad16 cells [4]. Also, rad26 rpb9 double mutants are moderately UV sensitive and rad16 rad26 rpb9 cells are more UV sensitive than rad1 cells that are entirely defective in NER. Rad26- and Rpb9-dependent TC-NER have been shown to have different efficiencies in different genes and in different regions of a gene. For slowly and moderately transcribed genes (e.g., URA3 and RPB2 genes), TC-NER appears to be accomplished primarily by the Rad26-dependent mechanism, except for a short region immediately downstream of the transcription start site [4,5]. In contrast, for highly transcribed genes (e.g., galactose induced GAL1-10), Rpb9 can play a larger role in TC-NER than Rad26 [4,6]. Rad26-dependent TC-NER can be active in certain region upstream of the transcription start site and the efficiency in the upstream regions can be as high as that in the gene coding region [4,6]. On the other hand, Rpb9-dependent TC-NER operates more effectively in the region downstream of the transcription start site than in the upstream region [4,6]. The initiation sites of Rad26- and Rpb9-dependent TC-NER in the galactose induced GAL1-10 gene appears to be determined by the upstream activating sequence (UAS) but not by the TATA element or local sequences (where the TC-NER starts to occur) [6–8]. However, both the UAS and the TATA sequences are essential for confining Rad26-dependent repair to the transcribed strand. Mutation of the TATA sequence, which greatly reduces transcription, or deletion of the TATA or mutation of the UAS, which results in undetectable transcription, causes a substantial level of Rad26-dependent repair to occur in both strands [7]. Rpb9-dependent repair only occurs in the transcribed strand and is efficient only in the presence of both TATA and UAS sequences (i.e., in the presence of substantial level of transcription) [7]. Also, Rad26 appears to contribute to a
substantial level of repair in both strands of a number of repressed genes (e.g., ADH2, GAL1-10 and PHO5) where no transcription can be detected by using traditional techniques [8]. However, induction of transcription of these genes results in the confinement of the Rad26-dependent repair to the transcribed strand. How Rad26 contribute to repair in both strands of UAS/TATA-disrupted or repressed genes remain to be understood. About 90% of RNAP II transcription initiation events in yeast represent transcriptional noise, and the specificity of transcription initiation is comparable to that of DNA-binding proteins and other biological processes [9]. It is therefore likely that “noise” transcription may occur in both strands of the UAS/TATA-disrupted or repressed genes, which may be enough for triggering Rad26-dependent, but not Rpb9dependent TC-NER. Induction of transcription from one strand of a gene may prevent “noise” transcription from the other strand (in opposite direction), resulting in the confinement of Rad26dependent repair in the transcribed strand. However, this scenario remains to be tested. RNAP II has an intrinsic capacity for transcription bypass of lesions by incorporation or misincorporation of nucleotides across the lesions [10]. Two yeast Rpb1 mutations, G730D (rpb1G730D) and E1103G (rpb1E1103G), have been shown to impair and enhance transcription bypass of CPDs, respectively [11]. The rpb1E1103G mutation was observed to increase UV resistance of RAD26+ but not rad26 cells, suggesting that the increased UV resistance is dependent on Rad26-dependent TC-NER [11]. It was therefore proposed that transcription bypass of a lesion may expose the lesion to TCNER proteins after their Rad26-dependent recruitment, and hence lesion exposure would be required for TC-NER [11,12]. However, it was later found that the increased UV resistance is independent of Rad26 or any other NER factors [13]. Also, instead of being required for TC-NER, the rpb1E1103G mutation was found to attenuate TCNER in rad26 and RAD26+ cells. It appears that transcription bypass of lesions enhances tolerance of cells to DNA lesions but attenuates Rad26-dependent and independent (Rpb9-dependent) TC-NER [13]. 4. Properties of Rad26 and its human homolog CSB Rad26 is the homolog of human CSB, which along with CSA is implicated in Cockayne syndrome [2]. Rad26 consists of 1085 amino acids organized into distinct domains (Fig. 1A). The central part of Rad26 contains seven conserved motifs that exist in many DNA and RNA helicases, which couple ATP hydrolysis to the unwinding of double-stranded DNA or RNA into its component single-strands. However, Rad26 is a DNA-dependent ATPase without a DNA helicase activity [14]. A leucine latch (LL) motif was recently identified in the N-terminus of the S. pombe Rhp26 (Fig. 1B), the homolog of S. cerevisiae Rad26 [15]. The LL autoinhibits the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. The C terminal domain (CTD) counteracts this autoinhibition. Both the LL and CTD of Rhp26 appear to be needed for its proper function in DNA repair in S. pombe cells, as deletion of the CTD or overexpression of the full-length or LL-deleted Rhp26 causes increased UV sensitivity [15]. Like the S. pombe Rhp26, human CSB also has an N-terminal region that autoinhibits its ATPase activity (Fig. 1C) [16]. The LL motif and CTD also exist in the S. cerevisiae Rad26 and they regulate the ATPase activity in a similar manner (Dong Wang, personal communications). How the LL and CTD affect TC-NER in the cell has not been directly analyzed. Upon treatment of yeast cells with various DNA damaging agents (H2 O2 , 4-nitroquinoline-1-oxide, methyl methanesulfonate and UV), Rad26 is phosphorylated primarily on serine 27 by the DNA damage checkpoint kinase Mec1, the homolog of human ATR [17]. The phosphorylation appears to enhance TC-NER to a certain
Please cite this article in press as: S. Li, Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.09.006
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Fig. 1. Domains of Rad26. A–C. Schematics of S. cerevisiae Rad26, S. pombe Rhp26 and human CSB. Alignment of motif sequences was done by using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/ clustalo). LL, leucine latch. NLS, nuclear localization signal. CTD, C-terminal domain. UBD, ubiquitin-binding domain.
extent. Serine 27 is located in the LL domain, which autoinhibits the ATPase activity of Rad26 (see above). However, the enhanced TC-NER may not be due to a change in the ATPase activity, as the serine 27 phosphorylation does not seem to significantly affect the ATPase activity [17]. Although they are highly homologous, Rad26 and CSB appear to have certain structural and behavioral differences. First, as mentioned above, elimination of Rad26 in otherwise wild type yeast cells does not cause detectable UV sensitivity [2], whereas human cells lacking CSB are moderately UV sensitive [18]. Second, CSB contains a C-terminal ubiquitin binding domain (UBD) (amino acids 1400–1428, Fig. 1C), which has been shown to be essential for CSB to function in TC-NER [19]. However, the yeast Rad26 lacks the UBD. Third, CSB is ubiquitinated and degraded by the proteasome mediated pathway in a late stage of the TC-NER process [20]. The Rad26 protein does not seem to be ubiquitinated or degraded during the TC-NER process in yeast [17]. Forth, CSB appears to loosely interact with the elongating RNAP II, but becomes more tightly bound following transcription arrest by DNA lesions [21,22]. To date, no direct interaction between Rad26 and RNAP II has been documented. Rad26 was shown to be recruited to the coding sequences of genes in a transcription-dependent but DNA-lesion-independent manner [23]. The recruitment is stimulated by methylation of histone H3 lysine 36, which is associated with active transcription. Finally, Rad26 has been shown to interact with Def1, a protein that is required for ubiquitination and proteolysis of RNAP II in response to DNA damage but plays no role in TC-NER [24]. To date, no homolog of the yeast Def1 has been identified in humans.
5. Other functions of Rad26 and CSB that may or may not be directly related to TC-NER 5.1. Recovery of RNA synthesis after UV irradiation Like human Cockayne syndrome cells [25], yeast cells lacking Rad26 have delayed recovery of RNA synthesis following UV irradiation [26]. The repressed RNA synthesis after UV irradiation may not only be due to blockage of transcription elongation by DNA lesions but also caused by repression of transcription initiation [27]. For example, both DNA lesions per se [28] and the NER machinery [29] may sequester transcription initiation factors (e.g., TFIID/TBP and TFIIH). Removal of DNA lesions is required but may not be sufficient for full recovery of RNA synthesis. Depletion of ELL, a TFIIH partner, does not seem to significantly affect TC-NER, as the depletion does not affect DNA synthesis associated with TC-NER [30].
However, depletion of ELL was shown to hinder recovery of RNA synthesis after UV irradiation [30]. 5.2. Transcription elongation Yeast cells lacking Rad26 have slower induction of certain inducible genes and, under certain conditions, are sensitive to 6azauracil, a base analogue that depletes cellular levels of the RNA precursors GTP and UTP, indicating that Rad26 plays a role in transcription elongation [31]. The transcription elongation defects of rad26 cells are synergistically enhanced by mutations of DST1, which encodes a general transcription elongation factor [31], and RAD2 [32]. In addition to catalyzing incision 3 to a lesion during NER, Rad2 also plays a role in transcription by interacting with and stabilizing TFIIH, a general transcription factor [32,33]. 5.3. Chromatin remodeling Rad26 is a member of the SWI2/SNF2 ATP-dependent chromatin remodeling protein family [34]. The S. pombe Rhp26 [15] and human CSB [35] have been shown to remodel chromatin structure in vitro. The chromatin remodeling activity may be required for TC-NER in vivo, as a mutant CSB that cannot remodel chromatin structure in vitro can only marginally rescue the UV resistance of human cells lacking CSB [36]. It was proposed that the chromatin remodeling activity may also remodel/destabilize lesion-stalled RNAP II complex, making the lesion accessible to the NER machinery. The S. cerevisiae Rad26 can also remodel chromatin structure in vitro (Dong Wang, personal communications). Overexpression of Rad26 in S. cerevisiae has been shown to increase repair of UV photoproducts in both the transcribed and nonstranscribed strand of a gene [37]. It was proposed that the increased repair may be due to increased remodeling of chromatin structure that leads to increased accessibility of the damaged DNA in chromatin for repair proteins. However, if and/or how the activity of chromatin remodeling is involved in TC-NER in yeast remains to be determined. 6. Repressors of TC-NER in cerevisiae 6.1. Rpb4/Rpb7 Among the 12 subunits (Rpb1-12) of RNAP II, Rpb4 and Rpb9 are not essential for cell viability. While Rpb9 facilitates TC-NER (see above), Rpb4 appears to repress TC-NER, as elimination of Rpb4 enhances TC-NER in RAD26+ cells (especially in stationary phase) and fully restores TC-NER in rad26 cells [4]. Rpb4 and Rpb7 form
Please cite this article in press as: S. Li, Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.09.006
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Fig. 2. Architecture of interactions between Spt4/5 and RNAP II. A. Locations of Spt4 and different domains of Spt5 on RNAP II. Spt4, the NGN domain of Spt5, Rpb1, Rbp2, Rbp4 and Rpb7 are shown in colors as indicated. Other subunits of RNAP II are shown in gray. Residues of RNAP II that cross-link to Spt5 are shown in pink. The likely locations of the Spt5 KOW domains are marked with red dashed-line ellipses. The acidic and C-terminal repeat (CTR) domains may not directly interact with RNAP II. B. Schematic cut-away view of the interaction architecture. The dashed line indicates the open clamp position observed in the absence of Rpb4/7. (C) Schematic showing different domains of Spt5. See [42] for more details.
a stalk structure (Fig. 2A), which is dissociable from the 10 subunit core RNAP II [38]. Association of the Rpb4/7 stalk with the core RNAP II “wedges” the clamp, composed of a region of Rpb1, to the closed conformation, resulting in a narrower central cleft of the polymerase (Fig 2B). The closed confirmation of RNAP II may restrict the access of the NER machinery to a lesion trapped in the transcription complex [4]. 6.2. Spt4/Spt5 The Spt4/Spt5 complex is a transcription elongation factor [39]. Elimination of Spt4 fully restores TC-NER in rad26 cells but does
not seem to significantly affect TC-NER in RAD26+ cells, indicating that Spt4 represses TC-NER only in the absence of Rad26 [40]. We found that the repression of TC-NER by Spt4 is accomplished through Spt5, by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with RNAP II [41]. Spt5 is a relatively large (1063 residue) protein containing multiple domains: the Nterminal acidic domain, the NGN domain, 5 KOW domains and the C-terminal repeat (CTR) domain, which contains 15 copies of a 6-amino acid sequence that can be phosphorylated by the Bur kinase domain (Fig. 2C) [39]. In vivo site-specific crosslinking studies showed that, through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7 [42]. Spt5 also interacts with Rpb1 and Rpb2,
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two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Spt4 does not directly interact with RNAP II, but may associate with RNAP II through interaction with the NGN domain of Spt5 (Fig. 2) [42,43]. Like elimination of Rpb4, but different from elimination of Spt4, deletion of the Spt5 KOW4-5 domains enhances TC-NER in RAD26+ cells and fully restores TC-NER in rad26 cells [42]. 6.3. RNAP II associated factor 1 complex (Paf1C) Paf1C is another transcription elongation factor [44]. It was reported that TC-NER in RAD26+ cells lacking Rtf1, one of the 5 subunits of Paf1C, was as inefficient as rad26 cells, suggesting that Paf1C plays an important role in facilitating TC-NER [45]. However, we found later that elimination of any of the 5 subunits of Paf1C causes marginal TC-NER deficiency in RAD26+ cells, but partially restores TC-NER in rad26 cells, indicating that Paf1C partially represses TC-NER in the absence of Rad26 [46]. Paf1C is recruited to RNAP II complex through interaction with the Spt5 CTR domain [46,47], which does not seem to directly interact with RNAP II [42]. Deletion of the Spt5 CTR also partially restores TC-NER in rad26 cells [41]. It is therefore likely that the repression of TC-NER by Paf1C may be achieved through and/or by coordination with the Spt5 CTR. Taken together, Rpb4 (in complex with Rpb7) and Spt5 appear to be strong TC-NER repressors. They can strongly repress TC-NER in the absence of Rad26 and moderately repress TC-NER in the presence of Rad26. On the other hand, Spt4 and Paf1C are weaker TC-NER repressors, as they can repress and partially repress TCNER, respectively, only in the absence but not in the presence of Rad26. Through direct interactions with Rpb4/Rpb7 and other domains of RNAP II, Spt5 appears to play a key role in coordinating the repression of TC-NER. Spt4 and Paf1C, by interacting with the NGN and CTR domains of Spt5, respectively, may play accessory roles in repressing TC-NER by further stabilizing the closed confirmation of the RNAP II complex [42]. Rad26 appears to be able to completely antagonize the accessory TC-NER repressors but can only partially alleviate the repression of TC-NER by the coordinated actions of Spt5 and Rpb4/Rpb7. 7. Possible ways for Rad26 to facilitate TC-NER In view of the fact that Rad26 can be entirely dispensable for TC-NER in the absence of a TC-NER repressor, it is unlikely that Rad26 directly recruits the NER machinery to a lesion site where RNAP II stalls. Instead, Rad26 may facilitate TC-NER indirectly, by counteracting the TC-NER repressors. The closed RNAP II complex stabilized by coordinated actions of Spt5 and other transcription elongation factors may be highly effective for transcription elongation but intrinsically repressive to TC-NER. One model is that the chromatin remodeling activity of Rad26 may remodel or ‘loosen’ the interactions of the TC-NER repressors with RNAP II, making a lesion trapped in the complex accessible to the NER machinery. RNAP II can either be stalled at or bypass a DNA lesion depending on the nucleotide(s) incorporated across the damaged template [11,48–50]. In view of the observations that transcription bypass of lesions attenuates TC-NER and efficient stalling of RNAP II may be required for efficient TC-NER [13], it is likely that a TC-NER signal can only be generated following insertion of certain nucleotide(s) across a lesion that leads to transcription stalling. Improper insertion of nucleotide(s) across a lesion that leads to transcription bypass will not elicit TC-NER. As the TC-NER repressors are transcription elongations factors, they may modulate the incorporation
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of nucleotides across lesions that lead to bypass of the lesions. Rad26 has been shown to modulate transcription across damaged templates [51,52]. CSB has been shown to push the RNAP II forward, such that an additional nucleotide is incorporated opposite a CPD [53]. An appealing alternative model is that Rad26 may facilitate incorporation of nucleotides across lesions that lead to stalling of RNAP II, thereby alleviating the inhibition of TC-NER by the repressors. This model and the aforementioned RNAP II complex remodeling model may not necessarily be mutually exclusive. The remodeling may affect the incorporation of nucleotides across a lesion and vise versa. Future work is needed to test these models. 8. Conflict of interest None Acknowledgements I thank Wentao Li for discussion of this minreview. This work was supported by National Science Foundation grant MCB1244019. References [1] D. Tatum, S. Li, Nucleotide Excision Repair in S. Cerevisiae, in Dna Repair - on the Pathways to Fixing DNA Damage and Errors, in: F. Storici (Ed.), InTech Open Access Publisher, Rijeka, Croatia, 2011, pp. 97–122. [2] A.J. van Gool, et al., RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6, EMBO J. 13 (22) (1994) 5361–5369. [3] R.A. Verhage, et al., Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair, Mol. Cell Biol. 16 (2) (1996) 496–502. [4] S. Li, M.J. Smerdon, Rpb4 and Rpb9 mediate subpathways of transcription-coupled DNA repair in Saccharomyces cerevisiae, EMBO J. 21 (21) (2002) 5921–5929. [5] M. Tijsterman, et al., Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. U. S. A. 94 (15) (1997) 8027–8032. [6] S. Li, M.J. Smerdon, Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes, J. Biol. Chem. 279 (14) (2004) 14418–14426. [7] S. Li, et al., Modulation of Rad26- and Rpb9-mediated DNA repair by different promoter elements, J. Biol. Chem. 281 (48) (2006) 36643–36651. [8] S. Li, et al., The roles of Rad1 and Rad26 in repairing repressed and actively transcribed genes in yeast, DNA Repair (Amst) 6 (11) (2007) 1596–1606. [9] K. Struhl, Transcriptional noise and the fidelity of initiation by RNA polymerase II, Nat. Struct. Mol. Biol. 14 (2) (2007) 103–105. [10] L. Xu, et al., Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis, DNA Repair (Amst) 19 (2014) 71–83. [11] C. Walmacq, et al., Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage, Mol. Cell 46 (1) (2012) 18–29. [12] T. Ellenberger, An arresting development in transcription, Mol. Cell 46 (1) (2012) 3–4. [13] W. Li, et al., Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair, Nucleic Acids Res. 42 (21) (2014) 13242–13253. [14] S.N. Guzder, et al., RAD26, the yeast homolog of human Cockayne’s syndrome group B gene, encodes a DNA-dependent ATPase, J. Biol. Chem. 271 (31) (1996) 18314–18317. [15] L. Wang, et al., Regulation of the Rhp26ERCC6/CSB chromatin remodeler by a novel conserved leucine latch motif, Proc. Natl. Acad. Sci. U. S. A. 111 (52) (2014) 18566–18571. [16] R.J. Lake, et al., UV-induced association of the CSB remodeling protein with chromatin requires ATP-dependent relief of N-terminal autorepression, Mol. Cell 37 (2) (2010) 235–246. [17] M. Taschner, et al., A role for checkpoint kinase-dependent Rad26 phosphorylation in transcription-coupled DNA repair in Saccharomyces cerevisiae, Mol. Cell Biol. 30 (2) (2010) 436–446. [18] H. de Waard, et al., Different effects of CSA and CSB deficiency on sensitivity to oxidative DNA damage, Mol. Cell Biol. 24 (18) (2004) 7941–7948. [19] R. Anindya, et al., A ubiquitin-binding domain in Cockayne syndrome B required for transcription-coupled nucleotide excision repair, Mol. Cell 38 (5) (2010) 637–648. [20] R. Groisman, et al., CSA-dependent degradation of CSB by the ubiquitin-proteasome pathway establishes a link between complementation factors of the Cockayne syndrome, Genes Dev. 20 (11) (2006) 1429–1434.
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Contents lists available at ScienceDirect
DNA Repair journal homepage: www.elsevier.com/locate/dnarepair
Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26 Shisheng Li ∗ Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, USA
a r t i c l e
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Article history: Available online xxx Keywords: Rad26 RNA polymerase II Saccharomyces cerevisiae Transcription coupled nucleotide excision repair Transcription elongation factors
a b s t r a c t Transcription coupled nucleotide excision repair (TC-NER) is believed to be triggered by an RNA polymerase stalled at a lesion in the transcribed strand of actively transcribed genes. Rad26, a DNA-dependent ATPase in the family of SWI2/SNF2 chromatin remodeling proteins, plays an important role in TC-NER in Saccharomyces cerevisiae. However, Rad26 is not solely responsible for TC-NER and Rpb9, a nonessential subunit of RNA polymerase II (RNAP II), is largely responsible for Rad26-independent TC-NER. The Rad26dependent and Rpb9-dependent TC-NER have different efficiencies in genes with different transcription levels and in different regions of a gene. Rad26 becomes entirely or partially dispensable for TC-NER in the absence of Rpb4, another nonessential subunit of RNAP II, or a number of transcription elongation factors (Spt4, Spt5 and the RNAP II associated factor complex). Rad26 may not be a true transcription-repair coupling factor that recruits the repair machinery to the damaged sites where RNAP II stalls. Rather, Rad26 may facilitate TC-NER indirectly, by antagonizing the action of TC-NER repressors that normally promote transcription elongation. The underlying mechanism of how Rad26 functions in TC-NER remains to be elucidated. © 2015 Elsevier B.V. All rights reserved.
Contents 1. 2. 3. 4. 5.
6.
7. 8.
Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 The involvement of Rad26 in TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Properties of Rad26 and its human homolog CSB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Other functions of Rad26 and CSB that may or may not be directly related to TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Recovery of RNA synthesis after UV irradiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.2. Transcription elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Chromatin remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Repressors of TC-NER in cerevisiae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 6.1. Rpb4/Rpb7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. Spt4/Spt5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.3. RNAP II associated factor 1 complex (Paf1C) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Possible ways for Rad26 to facilitate TC-NER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Prologue
∗ Correspondence address. Fax: +1 225 578 9895. E-mail address: [email protected]
My postdoc experience at Michael (Mick) Smerdon laboratory greatly influenced my research career. Mick is very passionate about how DNA repair takes place in chromatin. However, he is not the “my way or the highway” kind of person, and allows and encourages lab people to explore other avenues of research related
http://dx.doi.org/10.1016/j.dnarep.2015.09.006 1568-7864/© 2015 Elsevier B.V. All rights reserved.
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to DNA repair. He frequently forgets my and other people’s names but pays great attentions to experimental and data details. He is a firm believer that “bad data is worse than no data at all”. 2. Introduction Nucleotide excision repair (NER) is a highly conserved multistep process that removes bulky and/or helix-distorting DNA lesions, such as UV induced cyclobutane pyrimidine dimers (CPDs) [1] (also see elsewhere in this issue). Transcription coupled NER (TC-NER) is the NER subpathway that is dedicated to rapid removal of lesions in the transcribed strand of actively transcribed genes. Global genomic NER (GG-NER), on the other hand, refers to NER throughout the genome. The two NER subpathways rely on different proteins for recognition of DNA lesions but share common factors in the later steps of the repair process. In Saccharomyces cerevisiae, Rad26 plays an important role in TC-NER, whereas Rad7, Rad16 and Elc1 are specifically required for GG-NER. In this minireview, I will summarize findings about how Rad26 may be implicated in TC-NER. 3. The involvement of Rad26 in TC-NER Rad26 was found to play an important role in TC-NER in S. cerevisiae [2]. However, disruption of RAD26 in otherwise wild type yeast cells does not cause detectable UV sensitivity. rad16 rad26 or rad7 rad26 cells are more UV sensitive than the single rad7 or rad16 cells but not as UV sensitive as cells that are entirely defective in NER (e.g., rad14) [3]. The mild effect of Rad26 on UV sensitivity may be due to the fact that Rad26 is not solely responsible for TC-NER and the very efficient GG-NER in yeast may largely compensate for the loss of Rad26-dependent TC-NER for cell survival [1]. The Rad26-independent TC-NER was found to be dependent on Rpb9, a nonessential subunit of the 12 subunit (Rpb112) RNAP II [4]. Indeed, like rad16 rad26 cells, rad16 rpb9 cells are moderately more UV sensitive than rad16 cells [4]. Also, rad26 rpb9 double mutants are moderately UV sensitive and rad16 rad26 rpb9 cells are more UV sensitive than rad1 cells that are entirely defective in NER. Rad26- and Rpb9-dependent TC-NER have been shown to have different efficiencies in different genes and in different regions of a gene. For slowly and moderately transcribed genes (e.g., URA3 and RPB2 genes), TC-NER appears to be accomplished primarily by the Rad26-dependent mechanism, except for a short region immediately downstream of the transcription start site [4,5]. In contrast, for highly transcribed genes (e.g., galactose induced GAL1-10), Rpb9 can play a larger role in TC-NER than Rad26 [4,6]. Rad26-dependent TC-NER can be active in certain region upstream of the transcription start site and the efficiency in the upstream regions can be as high as that in the gene coding region [4,6]. On the other hand, Rpb9-dependent TC-NER operates more effectively in the region downstream of the transcription start site than in the upstream region [4,6]. The initiation sites of Rad26- and Rpb9-dependent TC-NER in the galactose induced GAL1-10 gene appears to be determined by the upstream activating sequence (UAS) but not by the TATA element or local sequences (where the TC-NER starts to occur) [6–8]. However, both the UAS and the TATA sequences are essential for confining Rad26-dependent repair to the transcribed strand. Mutation of the TATA sequence, which greatly reduces transcription, or deletion of the TATA or mutation of the UAS, which results in undetectable transcription, causes a substantial level of Rad26-dependent repair to occur in both strands [7]. Rpb9-dependent repair only occurs in the transcribed strand and is efficient only in the presence of both TATA and UAS sequences (i.e., in the presence of substantial level of transcription) [7]. Also, Rad26 appears to contribute to a
substantial level of repair in both strands of a number of repressed genes (e.g., ADH2, GAL1-10 and PHO5) where no transcription can be detected by using traditional techniques [8]. However, induction of transcription of these genes results in the confinement of the Rad26-dependent repair to the transcribed strand. How Rad26 contribute to repair in both strands of UAS/TATA-disrupted or repressed genes remain to be understood. About 90% of RNAP II transcription initiation events in yeast represent transcriptional noise, and the specificity of transcription initiation is comparable to that of DNA-binding proteins and other biological processes [9]. It is therefore likely that “noise” transcription may occur in both strands of the UAS/TATA-disrupted or repressed genes, which may be enough for triggering Rad26-dependent, but not Rpb9dependent TC-NER. Induction of transcription from one strand of a gene may prevent “noise” transcription from the other strand (in opposite direction), resulting in the confinement of Rad26dependent repair in the transcribed strand. However, this scenario remains to be tested. RNAP II has an intrinsic capacity for transcription bypass of lesions by incorporation or misincorporation of nucleotides across the lesions [10]. Two yeast Rpb1 mutations, G730D (rpb1G730D) and E1103G (rpb1E1103G), have been shown to impair and enhance transcription bypass of CPDs, respectively [11]. The rpb1E1103G mutation was observed to increase UV resistance of RAD26+ but not rad26 cells, suggesting that the increased UV resistance is dependent on Rad26-dependent TC-NER [11]. It was therefore proposed that transcription bypass of a lesion may expose the lesion to TCNER proteins after their Rad26-dependent recruitment, and hence lesion exposure would be required for TC-NER [11,12]. However, it was later found that the increased UV resistance is independent of Rad26 or any other NER factors [13]. Also, instead of being required for TC-NER, the rpb1E1103G mutation was found to attenuate TCNER in rad26 and RAD26+ cells. It appears that transcription bypass of lesions enhances tolerance of cells to DNA lesions but attenuates Rad26-dependent and independent (Rpb9-dependent) TC-NER [13]. 4. Properties of Rad26 and its human homolog CSB Rad26 is the homolog of human CSB, which along with CSA is implicated in Cockayne syndrome [2]. Rad26 consists of 1085 amino acids organized into distinct domains (Fig. 1A). The central part of Rad26 contains seven conserved motifs that exist in many DNA and RNA helicases, which couple ATP hydrolysis to the unwinding of double-stranded DNA or RNA into its component single-strands. However, Rad26 is a DNA-dependent ATPase without a DNA helicase activity [14]. A leucine latch (LL) motif was recently identified in the N-terminus of the S. pombe Rhp26 (Fig. 1B), the homolog of S. cerevisiae Rad26 [15]. The LL autoinhibits the ATPase and chromatin-remodeling activities of Rhp26 via its interaction with the core ATPase domain. The C terminal domain (CTD) counteracts this autoinhibition. Both the LL and CTD of Rhp26 appear to be needed for its proper function in DNA repair in S. pombe cells, as deletion of the CTD or overexpression of the full-length or LL-deleted Rhp26 causes increased UV sensitivity [15]. Like the S. pombe Rhp26, human CSB also has an N-terminal region that autoinhibits its ATPase activity (Fig. 1C) [16]. The LL motif and CTD also exist in the S. cerevisiae Rad26 and they regulate the ATPase activity in a similar manner (Dong Wang, personal communications). How the LL and CTD affect TC-NER in the cell has not been directly analyzed. Upon treatment of yeast cells with various DNA damaging agents (H2 O2 , 4-nitroquinoline-1-oxide, methyl methanesulfonate and UV), Rad26 is phosphorylated primarily on serine 27 by the DNA damage checkpoint kinase Mec1, the homolog of human ATR [17]. The phosphorylation appears to enhance TC-NER to a certain
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Fig. 1. Domains of Rad26. A–C. Schematics of S. cerevisiae Rad26, S. pombe Rhp26 and human CSB. Alignment of motif sequences was done by using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/ clustalo). LL, leucine latch. NLS, nuclear localization signal. CTD, C-terminal domain. UBD, ubiquitin-binding domain.
extent. Serine 27 is located in the LL domain, which autoinhibits the ATPase activity of Rad26 (see above). However, the enhanced TC-NER may not be due to a change in the ATPase activity, as the serine 27 phosphorylation does not seem to significantly affect the ATPase activity [17]. Although they are highly homologous, Rad26 and CSB appear to have certain structural and behavioral differences. First, as mentioned above, elimination of Rad26 in otherwise wild type yeast cells does not cause detectable UV sensitivity [2], whereas human cells lacking CSB are moderately UV sensitive [18]. Second, CSB contains a C-terminal ubiquitin binding domain (UBD) (amino acids 1400–1428, Fig. 1C), which has been shown to be essential for CSB to function in TC-NER [19]. However, the yeast Rad26 lacks the UBD. Third, CSB is ubiquitinated and degraded by the proteasome mediated pathway in a late stage of the TC-NER process [20]. The Rad26 protein does not seem to be ubiquitinated or degraded during the TC-NER process in yeast [17]. Forth, CSB appears to loosely interact with the elongating RNAP II, but becomes more tightly bound following transcription arrest by DNA lesions [21,22]. To date, no direct interaction between Rad26 and RNAP II has been documented. Rad26 was shown to be recruited to the coding sequences of genes in a transcription-dependent but DNA-lesion-independent manner [23]. The recruitment is stimulated by methylation of histone H3 lysine 36, which is associated with active transcription. Finally, Rad26 has been shown to interact with Def1, a protein that is required for ubiquitination and proteolysis of RNAP II in response to DNA damage but plays no role in TC-NER [24]. To date, no homolog of the yeast Def1 has been identified in humans.
5. Other functions of Rad26 and CSB that may or may not be directly related to TC-NER 5.1. Recovery of RNA synthesis after UV irradiation Like human Cockayne syndrome cells [25], yeast cells lacking Rad26 have delayed recovery of RNA synthesis following UV irradiation [26]. The repressed RNA synthesis after UV irradiation may not only be due to blockage of transcription elongation by DNA lesions but also caused by repression of transcription initiation [27]. For example, both DNA lesions per se [28] and the NER machinery [29] may sequester transcription initiation factors (e.g., TFIID/TBP and TFIIH). Removal of DNA lesions is required but may not be sufficient for full recovery of RNA synthesis. Depletion of ELL, a TFIIH partner, does not seem to significantly affect TC-NER, as the depletion does not affect DNA synthesis associated with TC-NER [30].
However, depletion of ELL was shown to hinder recovery of RNA synthesis after UV irradiation [30]. 5.2. Transcription elongation Yeast cells lacking Rad26 have slower induction of certain inducible genes and, under certain conditions, are sensitive to 6azauracil, a base analogue that depletes cellular levels of the RNA precursors GTP and UTP, indicating that Rad26 plays a role in transcription elongation [31]. The transcription elongation defects of rad26 cells are synergistically enhanced by mutations of DST1, which encodes a general transcription elongation factor [31], and RAD2 [32]. In addition to catalyzing incision 3 to a lesion during NER, Rad2 also plays a role in transcription by interacting with and stabilizing TFIIH, a general transcription factor [32,33]. 5.3. Chromatin remodeling Rad26 is a member of the SWI2/SNF2 ATP-dependent chromatin remodeling protein family [34]. The S. pombe Rhp26 [15] and human CSB [35] have been shown to remodel chromatin structure in vitro. The chromatin remodeling activity may be required for TC-NER in vivo, as a mutant CSB that cannot remodel chromatin structure in vitro can only marginally rescue the UV resistance of human cells lacking CSB [36]. It was proposed that the chromatin remodeling activity may also remodel/destabilize lesion-stalled RNAP II complex, making the lesion accessible to the NER machinery. The S. cerevisiae Rad26 can also remodel chromatin structure in vitro (Dong Wang, personal communications). Overexpression of Rad26 in S. cerevisiae has been shown to increase repair of UV photoproducts in both the transcribed and nonstranscribed strand of a gene [37]. It was proposed that the increased repair may be due to increased remodeling of chromatin structure that leads to increased accessibility of the damaged DNA in chromatin for repair proteins. However, if and/or how the activity of chromatin remodeling is involved in TC-NER in yeast remains to be determined. 6. Repressors of TC-NER in cerevisiae 6.1. Rpb4/Rpb7 Among the 12 subunits (Rpb1-12) of RNAP II, Rpb4 and Rpb9 are not essential for cell viability. While Rpb9 facilitates TC-NER (see above), Rpb4 appears to repress TC-NER, as elimination of Rpb4 enhances TC-NER in RAD26+ cells (especially in stationary phase) and fully restores TC-NER in rad26 cells [4]. Rpb4 and Rpb7 form
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Fig. 2. Architecture of interactions between Spt4/5 and RNAP II. A. Locations of Spt4 and different domains of Spt5 on RNAP II. Spt4, the NGN domain of Spt5, Rpb1, Rbp2, Rbp4 and Rpb7 are shown in colors as indicated. Other subunits of RNAP II are shown in gray. Residues of RNAP II that cross-link to Spt5 are shown in pink. The likely locations of the Spt5 KOW domains are marked with red dashed-line ellipses. The acidic and C-terminal repeat (CTR) domains may not directly interact with RNAP II. B. Schematic cut-away view of the interaction architecture. The dashed line indicates the open clamp position observed in the absence of Rpb4/7. (C) Schematic showing different domains of Spt5. See [42] for more details.
a stalk structure (Fig. 2A), which is dissociable from the 10 subunit core RNAP II [38]. Association of the Rpb4/7 stalk with the core RNAP II “wedges” the clamp, composed of a region of Rpb1, to the closed conformation, resulting in a narrower central cleft of the polymerase (Fig 2B). The closed confirmation of RNAP II may restrict the access of the NER machinery to a lesion trapped in the transcription complex [4]. 6.2. Spt4/Spt5 The Spt4/Spt5 complex is a transcription elongation factor [39]. Elimination of Spt4 fully restores TC-NER in rad26 cells but does
not seem to significantly affect TC-NER in RAD26+ cells, indicating that Spt4 represses TC-NER only in the absence of Rad26 [40]. We found that the repression of TC-NER by Spt4 is accomplished through Spt5, by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with RNAP II [41]. Spt5 is a relatively large (1063 residue) protein containing multiple domains: the Nterminal acidic domain, the NGN domain, 5 KOW domains and the C-terminal repeat (CTR) domain, which contains 15 copies of a 6-amino acid sequence that can be phosphorylated by the Bur kinase domain (Fig. 2C) [39]. In vivo site-specific crosslinking studies showed that, through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7 [42]. Spt5 also interacts with Rpb1 and Rpb2,
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two largest subunits of RNAP II, at the clamp, protrusion and wall domains. These interactions may lock the clamp to the closed conformation and enclose the DNA being transcribed in the central cleft of RNAP II. Spt4 does not directly interact with RNAP II, but may associate with RNAP II through interaction with the NGN domain of Spt5 (Fig. 2) [42,43]. Like elimination of Rpb4, but different from elimination of Spt4, deletion of the Spt5 KOW4-5 domains enhances TC-NER in RAD26+ cells and fully restores TC-NER in rad26 cells [42]. 6.3. RNAP II associated factor 1 complex (Paf1C) Paf1C is another transcription elongation factor [44]. It was reported that TC-NER in RAD26+ cells lacking Rtf1, one of the 5 subunits of Paf1C, was as inefficient as rad26 cells, suggesting that Paf1C plays an important role in facilitating TC-NER [45]. However, we found later that elimination of any of the 5 subunits of Paf1C causes marginal TC-NER deficiency in RAD26+ cells, but partially restores TC-NER in rad26 cells, indicating that Paf1C partially represses TC-NER in the absence of Rad26 [46]. Paf1C is recruited to RNAP II complex through interaction with the Spt5 CTR domain [46,47], which does not seem to directly interact with RNAP II [42]. Deletion of the Spt5 CTR also partially restores TC-NER in rad26 cells [41]. It is therefore likely that the repression of TC-NER by Paf1C may be achieved through and/or by coordination with the Spt5 CTR. Taken together, Rpb4 (in complex with Rpb7) and Spt5 appear to be strong TC-NER repressors. They can strongly repress TC-NER in the absence of Rad26 and moderately repress TC-NER in the presence of Rad26. On the other hand, Spt4 and Paf1C are weaker TC-NER repressors, as they can repress and partially repress TCNER, respectively, only in the absence but not in the presence of Rad26. Through direct interactions with Rpb4/Rpb7 and other domains of RNAP II, Spt5 appears to play a key role in coordinating the repression of TC-NER. Spt4 and Paf1C, by interacting with the NGN and CTR domains of Spt5, respectively, may play accessory roles in repressing TC-NER by further stabilizing the closed confirmation of the RNAP II complex [42]. Rad26 appears to be able to completely antagonize the accessory TC-NER repressors but can only partially alleviate the repression of TC-NER by the coordinated actions of Spt5 and Rpb4/Rpb7. 7. Possible ways for Rad26 to facilitate TC-NER In view of the fact that Rad26 can be entirely dispensable for TC-NER in the absence of a TC-NER repressor, it is unlikely that Rad26 directly recruits the NER machinery to a lesion site where RNAP II stalls. Instead, Rad26 may facilitate TC-NER indirectly, by counteracting the TC-NER repressors. The closed RNAP II complex stabilized by coordinated actions of Spt5 and other transcription elongation factors may be highly effective for transcription elongation but intrinsically repressive to TC-NER. One model is that the chromatin remodeling activity of Rad26 may remodel or ‘loosen’ the interactions of the TC-NER repressors with RNAP II, making a lesion trapped in the complex accessible to the NER machinery. RNAP II can either be stalled at or bypass a DNA lesion depending on the nucleotide(s) incorporated across the damaged template [11,48–50]. In view of the observations that transcription bypass of lesions attenuates TC-NER and efficient stalling of RNAP II may be required for efficient TC-NER [13], it is likely that a TC-NER signal can only be generated following insertion of certain nucleotide(s) across a lesion that leads to transcription stalling. Improper insertion of nucleotide(s) across a lesion that leads to transcription bypass will not elicit TC-NER. As the TC-NER repressors are transcription elongations factors, they may modulate the incorporation
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of nucleotides across lesions that lead to bypass of the lesions. Rad26 has been shown to modulate transcription across damaged templates [51,52]. CSB has been shown to push the RNAP II forward, such that an additional nucleotide is incorporated opposite a CPD [53]. An appealing alternative model is that Rad26 may facilitate incorporation of nucleotides across lesions that lead to stalling of RNAP II, thereby alleviating the inhibition of TC-NER by the repressors. This model and the aforementioned RNAP II complex remodeling model may not necessarily be mutually exclusive. The remodeling may affect the incorporation of nucleotides across a lesion and vise versa. Future work is needed to test these models. 8. Conflict of interest None Acknowledgements I thank Wentao Li for discussion of this minreview. This work was supported by National Science Foundation grant MCB1244019. References [1] D. Tatum, S. Li, Nucleotide Excision Repair in S. Cerevisiae, in Dna Repair - on the Pathways to Fixing DNA Damage and Errors, in: F. Storici (Ed.), InTech Open Access Publisher, Rijeka, Croatia, 2011, pp. 97–122. [2] A.J. van Gool, et al., RAD26, the functional S. cerevisiae homolog of the Cockayne syndrome B gene ERCC6, EMBO J. 13 (22) (1994) 5361–5369. [3] R.A. Verhage, et al., Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair, Mol. Cell Biol. 16 (2) (1996) 496–502. [4] S. Li, M.J. Smerdon, Rpb4 and Rpb9 mediate subpathways of transcription-coupled DNA repair in Saccharomyces cerevisiae, EMBO J. 21 (21) (2002) 5921–5929. [5] M. Tijsterman, et al., Transitions in the coupling of transcription and nucleotide excision repair within RNA polymerase II-transcribed genes of Saccharomyces cerevisiae, Proc. Natl. Acad. Sci. U. S. A. 94 (15) (1997) 8027–8032. [6] S. Li, M.J. Smerdon, Dissecting transcription-coupled and global genomic repair in the chromatin of yeast GAL1-10 genes, J. Biol. Chem. 279 (14) (2004) 14418–14426. [7] S. Li, et al., Modulation of Rad26- and Rpb9-mediated DNA repair by different promoter elements, J. Biol. Chem. 281 (48) (2006) 36643–36651. [8] S. Li, et al., The roles of Rad1 and Rad26 in repairing repressed and actively transcribed genes in yeast, DNA Repair (Amst) 6 (11) (2007) 1596–1606. [9] K. Struhl, Transcriptional noise and the fidelity of initiation by RNA polymerase II, Nat. Struct. Mol. Biol. 14 (2) (2007) 103–105. [10] L. Xu, et al., Molecular basis of transcriptional fidelity and DNA lesion-induced transcriptional mutagenesis, DNA Repair (Amst) 19 (2014) 71–83. [11] C. Walmacq, et al., Mechanism of translesion transcription by RNA polymerase II and its role in cellular resistance to DNA damage, Mol. Cell 46 (1) (2012) 18–29. [12] T. Ellenberger, An arresting development in transcription, Mol. Cell 46 (1) (2012) 3–4. [13] W. Li, et al., Transcription bypass of DNA lesions enhances cell survival but attenuates transcription coupled DNA repair, Nucleic Acids Res. 42 (21) (2014) 13242–13253. [14] S.N. Guzder, et al., RAD26, the yeast homolog of human Cockayne’s syndrome group B gene, encodes a DNA-dependent ATPase, J. Biol. Chem. 271 (31) (1996) 18314–18317. [15] L. Wang, et al., Regulation of the Rhp26ERCC6/CSB chromatin remodeler by a novel conserved leucine latch motif, Proc. Natl. Acad. Sci. U. S. A. 111 (52) (2014) 18566–18571. [16] R.J. Lake, et al., UV-induced association of the CSB remodeling protein with chromatin requires ATP-dependent relief of N-terminal autorepression, Mol. Cell 37 (2) (2010) 235–246. [17] M. Taschner, et al., A role for checkpoint kinase-dependent Rad26 phosphorylation in transcription-coupled DNA repair in Saccharomyces cerevisiae, Mol. Cell Biol. 30 (2) (2010) 436–446. [18] H. de Waard, et al., Different effects of CSA and CSB deficiency on sensitivity to oxidative DNA damage, Mol. Cell Biol. 24 (18) (2004) 7941–7948. [19] R. Anindya, et al., A ubiquitin-binding domain in Cockayne syndrome B required for transcription-coupled nucleotide excision repair, Mol. Cell 38 (5) (2010) 637–648. [20] R. Groisman, et al., CSA-dependent degradation of CSB by the ubiquitin-proteasome pathway establishes a link between complementation factors of the Cockayne syndrome, Genes Dev. 20 (11) (2006) 1429–1434.
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Please cite this article in press as: S. Li, Transcription coupled nucleotide excision repair in the yeast Saccharomyces cerevisiae: The ambiguous role of Rad26, DNA Repair (2015), http://dx.doi.org/10.1016/j.dnarep.2015.09.006