The C-terminal Repeat Domain of Spt5 Plays an Important Role in Suppression of Rad26-independent Transcription Coupled Repair

Abstract

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 rad26⌬ 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 rad26⌬ 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. Nucleotide excision repair (NER)2 is a conserved DNA repair mechanism capable of removing a variety of helix-distorting lesions, such as UV-induced cyclobutane pyrimidine dimers (CPDs) (1). NER can be grouped into two pathways: global genomic repair (GGR), which refers to repair throughout the genome, and transcription coupled repair (TCR), which refers to a repair mechanism that is dedicated to the transcribed strand of actively transcribed genes (2). In the yeast Saccharomyces cerevisiae, Rad7, Rad16 (3), and Elc1 (4) are specifically required for GGR, but dispensable for TCR. Rad7 and Rad16 form a complex that binds specifically to UV-damaged DNA in an ATP-dependent manner and has DNA-dependent ATPase activity (5). Elc1 has been shown to be a component of a ubiquitin ligase that contains Rad7 and Rad16, and is responsible for regulating the levels of Rad4 protein in response to UV damage (6, 7). It has also been suggested that Elc1 is a component of another ubiquitin ligase complex, which contains Ela1, Cul3, and Roc1 but not Rad7 and Rad16 (8,9). The role of Elc1 in GGR may not be subsidiary to that of Rad7 and Rad16 (4).The mechanistic details of TCR are relatively well understood in Escherichia coli. The transcription repair coupling factor Mfd targets the transcribed strand for repair by recognizing a stalled RNA polymerase and actively recruiting the NER machinery to the transcription blocking lesion as it dissociates the stalled RNA polymerase (10). Conversely, the TCR mechanisms in eukaryotes appear to be extremely complicated (for reviews, see Refs. 11 and 12). In mammalian cells, Cockayne syndrome group A (CSA) and B (CSB) proteins are specifically required for TCR, but dispensable for GGR (13-16). Like its human homolog CSB, the yeast Rad26 plays an important role in TCR but is dispensable for GGR (17). Both human CSB (18) ...

Target DNA Sequence Directly Regulates the Frequency of Activation-Induced Deaminase-Dependent Mutations

“…Spt5 has been shown to interact with AID (34), and its genome localization pattern is highly correlative to that of RNAPII in the in vitro cytokine-activated B cells (34). Apart from associating with RNAPII paused at the promoter proximal region, Spt5 links RNAPII to other factors such as splicing factors, capping enzyme, and transcription coupled repair factors (69)(70)(71)(72)(73). Thus, it remains to be determined how Spt5 contributes to RNAPII pausing at S regions.…”

The Spt5 C-Terminal Region Recruits Yeast 3′ RNA Cleavage Factor I

“…Deletion of the Spt5 CTR in yeast is not lethal (16,45,89) but leads to sensitivity to 6-AU and a slow-growth phenotype at 16°C (45,89). The CTR deletion is synthetically lethal with the deletion of the gene for the Pol II CTD kinase Ctk1 (45).…”

“…In human cells, CTR phosphorylation by P-TEFb converts Spt5 from a negative to a positive elongation factor (87). The Spt5 CTR may also play a role in the suppression of transcriptioncoupled nucleotide excision repair in yeast (16).…”

“…Spt5 copurifies with over 90 yeast proteins that are involved in transcription elongation, RNA processing, transcription termination, and mRNA export (44). Spt4/5 interacts with the histone chaperone Spt6 to modulate chromatin structure (24,78) Deletion of the Spt5 CTR in yeast is not lethal (16,45,89) but leads to sensitivity to 6-AU and a slow-growth phenotype at 16°C (45, 89). The CTR deletion is synthetically lethal with the deletion of the gene for the Pol II CTD kinase Ctk1 (45).…”

The Transcription Elongation Factor Bur1-Bur2 Interacts with Replication Protein A and Maintains Genome Stability during Replication Stress

“…Bur1-Bur2 is required for monoubiquitylation of histone H2B at Lys-123 by Rad6, for trimethylation of histone H3 at Lys-4 by Set1, and for methylation of histone H3 at Lys-36 by Set2 (10 -12). Bur1-Bur2 function is further required for recruitment of the Paf1 complex by phosphorylation of the transcription elongation factor Spt5 (6,13), which was recently shown to be a Rad26-independent suppressor of transcription-coupled repair (14).…”