Spt5, a transcription elongation factor, and Rpb4, a subunit of RNA polymerase II (RNAP II) that forms a subcomplex with Rpb7, play important roles in transcription elongation and repression of transcription coupled DNA repair (TCR) in eukaryotic cells. How Spt5 physically interacts with RNAP II, and if and/or how Spt5 and Rpb4/7 coordinate to achieve the distinctive functions have been enigmatic. By site-specific incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine, a photoreactive cross-linker, we mapped interactions between Spt5 and RNAP II in Saccharomyces cerevisiae. Through its KOW4-5 domains, Spt5 extensively interacts with Rpb4/7. Spt5 also interacts with Rpb1 and Rpb2, 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. Deletion of Spt5 KOW4-5 domains decreases transcription elongation and derepresses TCR. Our findings suggest that Spt5 is a key coordinator for holding the RNAP II complex in a closed conformation that is highly competent for transcription elongation but repressive to TCR.

Global genomic repair (GGR) and transcription coupled repair (TCR) are two pathways of nucleotide excision repair (NER) that differ in the damage recognition step. How NER factors, especially GGR factors, access DNA damage in the chromatin of eukaryotic cells has been poorly understood. Dot1, a histone methyltransferase required for methylation of histone H3 lysine 79 (H3K79), has been shown to confer yeast cells with resistance to DNA-damaging agents and play a role in activation of DNA damage checkpoints. Here, we show that Dot1 and H3K79 methylation are required for GGR in both nucleosomal core regions and internucleosomal linker DNA, but play no role in TCR. H3K79 trimethylation contributes to but is not absolutely required for GGR, and lower levels of H3K79 methylation (mono- and dimethylation) also promote GGR. Our results also indicate that the roles of Dot1 and H3K79 methylation in GGR are not achieved by either activating DNA damage checkpoints or regulating the expression of the GGR-specific factor Rad16. Rather, the methylated H3K79 may serve as a docking site for the GGR machinery on the chromatin. Our studies identified a novel GGR-specific NER factor and unveiled the critical link between a covalent histone modification and GGR.

In eukaryotic cells, transcription coupled nucleotide excision repair (TCR) is believed to be initiated by RNA polymerase II (Pol II) stalled at a lesion in the transcribed strand of a gene. Rad26, the yeast homolog of the human Cockayne syndrome group B (CSB) protein, plays an important role in TCR. Spt4, a transcription elongation factor that forms a complex with Spt5, has been shown to suppress TCR in rad26Delta cells. Here we present evidence that Spt4 indirectly suppresses Rad26-independent TCR by protecting Spt5 from degradation and stabilizing the interaction of Spt5 with Pol II. We further found that the C-terminal repeat (CTR) domain of Spt5, which is dispensable for cell viability and is not involved in interactions with Spt4 and Pol II, plays an important role in the suppression. The Spt5 CTR is phosphorylated by the Bur kinase. Inactivation of the Bur kinase partially alleviates TCR in rad26Delta cells. We propose that the Spt5 CTR suppresses Rad26-independent TCR by serving as a platform for assembly of a multiple protein suppressor complex that is associated with Pol II. Phosphorylation of the Spt5 CTR by the Bur kinase may facilitate the assembly of the suppressor complex.

Histones are highly alkaline proteins that package and order the DNA into chromatin in eukaryotic cells. Nucleotide excision repair (NER) is a conserved multistep reaction that removes a wide range of generally bulky and/or helix-distorting DNA lesions. Although the core biochemical mechanism of NER is relatively well known, how cells detect and repair lesions in diverse chromatin environments is still under intensive research. As with all DNA-related processes, the NER machinery must deal with the presence of organized chromatin and the physical obstacles it presents. A huge catalogue of posttranslational histone modifications has been documented. Although a comprehensive understanding of most of these modifications is still lacking, they are believed to be important regulatory elements for many biological processes, including DNA replication and repair, transcription and cell cycle control. Some of these modifications, including acetylation, methylation, phosphorylation and ubiquitination on the four core histones (H2A, H2B, H3 and H4) or the histone H2A variant H2AX, have been found to be implicated in different stages of the NER process. This review will summarize our recent understanding in this area.

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 polymerase 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 II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating 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 elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.