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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 Rad26-dependent 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. [ABSTRACT FROM AUTHOR]

Rpb9, a non-essential subunit of RNA polymerase H, mediates a transcription-coupled repair (TCR) sub-pathway in Saccharomyces cerevisiae. This subpathway initiates at the same upstream site as the previously identified Rad26 subpathway. However, the Rpb9 subpathway operates more effectively in the coding region than in the region upstream of the transcription start site, whereas the Rad26 subpathway operates equally in the two regions. Rpb4, another non-essential subunit of RNA polymerase II, plays a dual role in regulating the two subpathways, suppressing the Rpb9 subpathway and facilitating the Rad26 subpathway. Simultaneous deletion of RPB9 and RAD26 genes completely abolishes TCR in both the coding and upstream regions, indicating that no other TCR subpathway exists in RNA polymerase II- transcribed genes. [ABSTRACT FROM AUTHOR]

Abstract Transcription coupled repair (TC-NER) is a subpathway of nucleotide excision repair triggered by stalling of RNA polymerase at DNA lesions. It has been suspected that transcriptional misincorporations of certain nucleotides opposite lesions that result in irreversible transcription stalling might be important for TC-NER. However, the spectra of nucleotide misincorporations opposite UV photoproducts and how they are implicated in transcriptional stalling and TC-NER in the cell remain unknown. Rad26, a low abundant yeast protein, and its human homolog CSB have been proposed to facilitate TC-NER in part by positioning and stabilizing stalling of RNA polymerase II (RNAPII) at DNA lesions. Here, we found that substantial AMPs but no other nucleotides are transcriptionally misincoporated and extended opposite UV photoproducts and adjacent bases in Saccharomyces cerevisiae. Rad26 does not significantly affect either the misincorporation or extension of AMPs. At normally low or moderately increased levels, Rad26 promotes error-free transcriptional bypass and TC-NER of UV photoproducts. However, Rad26 completely loses these functions when it is overexpressed to ~ 1/3 the level of RNAPII molecules. Also, Rad26 does not directly displace RNAPII but constitutively evicts Spt5, a key transcription elongation factor and TC-NER repressor, from the chromatin. Our results indicate that transcriptional nucleotide misincorporation is not implicated in TC-NER, and moderate eviction of Spt5 and promotion of error-free transcriptional bypass of DNA lesions by Rad26 facilitates TC-NER. Graphical Abstract Unlabelled Image Highlights • Rad26 was proposed to facilitate TC-NER in part by positioning and stabilizing RNAPII at DNA lesions. • Only AMPs are transcriptionally misincoporated and extended opposite UV photoproducts in the cell. • Only at low or moderate levels can Rad26 promote transcriptional bypass of UV photoproducts and TC-NER. • Rad26 does not directly displace RNAPII but evicts Spt5 from the chromatin. • Rad26 facilitates TC-NER by moderately evicting Spt5 and promoting transcriptional lesion bypass. [ABSTRACT FROM AUTHOR]

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA poly- merase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol Il-associated factor 1 complex (PafiC) has been shown to func- tion in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that PafiC plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooper- ating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elon- gation complex, which is stabilized by the coordinated interac- tions of Rpb4-Rpb7, Spt4-Spt5, and PafiC with each other and with the core Pol II. We also found that PafiC facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of his- tone H2B lysine 123 by Brel, which in turn permits di- and trim- ethylation of histone H3 lysine 79 by Doti. To our best knowl- edge, among the NER-modulating factors documented so far, PafiC appears to have the most diverse functions in different NER pathways or subpathways. [ABSTRACT FROM AUTHOR]

Abstract: Transcription coupled repair (TCR) is a nucleotide excision repair (NER) pathway that is dedicated to repair in the transcribed strand of an active gene. The genome overall NER is called global genomic repair (GGR). Elc1, the yeast homolog of the mammalian elongation factor elongin C, has been shown to be a component of a ubiquitin ligase complex that contains Rad7 and Rad16, two factors that are specifically required for GGR. Elc1 has also been suggested to be present in another ubiquitin ligase complex that lacks Rad7 and Rad16 and is involved in UV-induced ubiquitylation and subsequent degradation of RNA polymerase II. Here we show that elc1 deletion increases UV sensitivity of TCR-deficient cells but does not affect the UV sensitivity of otherwise wild type and GGR-deficient cells. Cells deleted for elc1 show normal NER in the transcribed strand of an active gene but have no detectable NER in the non-transcribed strand. Elc1 does not affect UV-induced mutagenesis when TCR is operative, but plays an important role in preventing the mutagenesis if TCR is defective. Furthermore, the levels of Rad7 and Rad16 proteins are not significantly decreased in elc1 cells, and overexpression of Rad7 and Rad16 individually or simultaneously in elc1 cells does not restore repair in the non-transcribed strand of an active gene. Our results suggest that Elc1 has no function in TCR but plays an important role in GGR. Furthermore, the role of Elc1 in GGR may not be subsidiary to that of Rad7 and Rad16. [Copyright &y& Elsevier]

Abstract: Nucleotide excision repair (NER) is a conserved DNA repair mechanism capable of removing a variety of helix-distorting DNA lesions. A specialized NER pathway, called transcription coupled NER (TC-NER), refers to preferential repair in the transcribed strand of an actively transcribed gene. To be distinguished from TCR-NER, the genome-wide NER process is termed as global genomic NER (GG-NER). In Saccharomyces cerevisiae, GG-NER is dependent on Rad7, whereas TC-NER is mediated by Rad26, the homolog of the human Cockayne syndrome group B protein, and by Rpb9, a non-essential subunit of RNA polymerase II. Tfb5, the tenth subunit of the transcription/repair factor TFIIH, is implicated in one group of the human syndrome trichothiodystrophy. Here, we show that Tfb5 plays different roles in different NER pathways in yeast. No repair takes place in the non-transcribed strand of a gene in tfb5 cells, or in both strands of a gene in rad26 rpb9 tfb5 cells, indicating that Tfb5 is essential for GG-NER. However, residual repair occurs in the transcribed strand of a gene in tfb5 cells, suggesting that Tfb5 is important, but not absolutely required for TC-NER. Interestingly, substantial repair occurs in the transcribed strand of a gene in rad7 tfb5 and rad7 rpb9 tfb5 cells, indicating that, in the absence of GG-NER, Tfb5 is largely dispensable for Rad26 mediated TC-NER. Furthermore, we show that no repair takes place in the transcribed strand of a gene in rad7 rad26 tfb5 cells, suggesting that Tfb5 is required for Rpb9 mediated TC-NER. Taken together, our results indicate that Tfb5 is partially dispensable for Rad26 mediated TC-NER, especially in GG-NER deficient cells. However, this TFIIH subunit is required for other NER pathways. [Copyright &y& Elsevier]