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 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.
Highlights
MCB-0745229. 1 To whom correspondence should be addressed
We further found that the C-terminal repeat (CTR) domain of Spt5, which contains 15 copies of a 6-amino acid sequence that can be phosphorylated by the Bur kinase, plays an important role in suppressing Rad26-independent transcription coupled nucleotide excision repair (TCR)
We show that the nonessential CTR domain of Spt5 plays an important role in suppression of Rad26-independent TCR
Summary
MATa ura trp leu2⌬1 his3⌬200 pep4::HIS3 prb1⌬1.6R can As BJ5465, but rad7⌬ rad26⌬ As BJ5465, but rad7⌬ rad26⌬ spt4::LEU2 As BJ5465, but rad7⌬ rad26⌬ (SPT4-3ϫFLAG) As BJ5465, but (SPT5-3ϫFLAG) As BJ5465, but rad7::URA3 (SPT5-3ϫFLAG) As BJ5465, but rad7⌬ rad26⌬ (SPT5-3ϫFLAG) As BJ5465, but spt4::LEU2 (SPT5-3ϫFLAG) As BJ5465, but rad7⌬ spt4::LEU2 (SPT5-3ϫFLAG) As BJ5465, but rad7⌬ rad26⌬ spt4::LEU2 (SPT5-3ϫFLAG) As CR18, butpGAL-SPT5͔ As CR78, butpGAL-SPT5͔ As BJ5465, butpGAL-SPT5͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/CTR⌬͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/641-1063⌬͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/422-1063⌬͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/1–244⌬͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/1–421⌬͔ As CR18, but spt5::KanMXpRS416-SPT5, pSPT5/1–640⌬͔ As BD56, butpRS416-SPT5͔ removed As BD57, butpRS416-SPT5͔ removed. Spt194 combined with an spt mutation leads to synthetic lethality [39]. Unlike spt4⌬, the spt194 mutation does not suppress UV sensitivity of rad16⌬ rad26⌬ cells [38]. This observation led to the proposition that, unlike Spt, Spt may not play a role in suppressing Rad26-independent TCR or that, despite the shared phenotypes with spt4⌬, the specific spt194 mutation may not lead to a defect in the suppression. We further found that the C-terminal repeat (CTR) domain of Spt, which contains 15 copies of a 6-amino acid sequence that can be phosphorylated by the Bur kinase, plays an important role in suppressing Rad26-independent TCR
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