Structural basis of human transcription–DNA repair coupling

AbstractTranscription-coupled DNA repair removes bulky DNA lesions from the genome1,2 and protects cells against ultraviolet (UV) irradiation3. Transcription-coupled DNA repair begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CSB, the E3 ubiquitin ligase, CRL4CSA and UV-stimulated scaffold protein A (UVSSA)3. Here we provide five high-resolution structures of Pol II transcription complexes containing human transcription-coupled DNA repair factors and the elongation factors PAF1 complex (PAF) and SPT6. Together with biochemical and published3,4 data, the structures provide a model for transcription–repair coupling. Stalling of Pol II at a DNA lesion triggers replacement of the elongation factor DSIF by CSB, which binds to PAF and moves upstream DNA to SPT6. The resulting elongation complex, ECTCR, uses the CSA-stimulated translocase activity of CSB to pull on upstream DNA and push Pol II forward. If the lesion cannot be bypassed, CRL4CSA spans over the Pol II clamp and ubiquitylates the RPB1 residue K1268, enabling recruitment of TFIIH to UVSSA and DNA repair. Conformational changes in CRL4CSA lead to ubiquitylation of CSB and to release of transcription-coupled DNA repair factors before transcription may continue over repaired DNA.

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Regulation of P-TEFb Elongation Complex Activity by CDK9 Acetylation

Molecular and Cellular Biology ◽

10.1128/mcb.00857-06 ◽

2007 ◽

Vol 27 (13) ◽

pp. 4641-4651 ◽

Cited By ~ 40

Author(s):

Junjiang Fu ◽

Ho-Geun Yoon ◽

Jun Qin ◽

Jiemin Wong

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Elongation Complex ◽

Complex Activity ◽

Interacting Protein ◽

Pol Ii ◽

Carboxy Terminal

ABSTRACT P-TEFb, comprised of CDK9 and a cyclin T subunit, is a global transcriptional elongation factor important for most RNA polymerase II (pol II) transcription. P-TEFb facilitates transcription elongation in part by phosphorylating Ser2 of the heptapeptide repeat of the carboxy-terminal domain (CTD) of the largest subunit of pol II. Previous studies have shown that P-TEFb is subjected to negative regulation by forming an inactive complex with 7SK small RNA and HEXIM1. In an effort to investigate the molecular mechanism by which corepressor N-CoR mediates transcription repression, we identified HEXIM1 as an N-CoR-interacting protein. This finding led us to test whether the P-TEFb complex is regulated by acetylation. We demonstrate that CDK9 is an acetylated protein in cells and can be acetylated by p300 in vitro. Through both in vitro and in vivo assays, we identified lysine 44 of CDK9 as a major acetylation site. We present evidence that CDK9 is regulated by N-CoR and its associated HDAC3 and that acetylation of CDK9 affects its ability to phosphorylate the CTD of pol II. These results suggest that acetylation of CDK9 is an important posttranslational modification that is involved in regulating P-TEFb transcriptional elongation function.

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Structural basis of transcriptional stalling and bypass of abasic DNA lesion by RNA polymerase II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1722050115 ◽

2018 ◽

Vol 115 (11) ◽

pp. E2538-E2545 ◽

Cited By ~ 19

Author(s):

Wei Wang ◽

Celine Walmacq ◽

Jenny Chong ◽

Mikhail Kashlev ◽

Dong Wang

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Lesions ◽

Structural Motif ◽

Structural Basis ◽

Dependent Manner ◽

Abasic Site ◽

Structural Explanation ◽

Pol Ii ◽

Abasic Sites

Abasic sites are among the most abundant DNA lesions and interfere with DNA replication and transcription, but the mechanism of their action on transcription remains unknown. Here we applied a combined structural and biochemical approach for a comprehensive investigation of how RNA polymerase II (Pol II) processes an abasic site, leading to slow bypass of lesion. Encounter of Pol II with an abasic site involves two consecutive slow steps: insertion of adenine opposite a noninstructive abasic site (the A-rule), followed by extension of the 3′-rAMP with the next cognate nucleotide. Further studies provided structural insights into the A-rule: ATP is slowly incorporated into RNA in the absence of template guidance. Our structure revealed that ATP is bound to the Pol II active site, whereas the abasic site is located at an intermediate state above the Bridge Helix, a conserved structural motif that is cirtical for Pol II activity. The next extension step occurs in a template-dependent manner where a cognate substrate is incorporated, despite at a much slower rate compared with nondamaged template. During the extension step, neither the cognate substrate nor the template base is located at the canonical position, providing a structural explanation as to why this step is as slow as the insertion step. Taken together, our studies provide a comprehensive understanding of Pol II stalling and bypass of the abasic site in the DNA template.

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Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415186112 ◽

2015 ◽

Vol 112 (5) ◽

pp. E410-E419 ◽

Cited By ~ 27

Author(s):

Celine Walmacq ◽

Lanfeng Wang ◽

Jenny Chong ◽

Kathleen Scibelli ◽

Lucyna Lubkowska ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Active Site ◽

Dna Lesions ◽

Abasic Site ◽

Pol Ii ◽

Dna Lesion ◽

Trigger Loop ◽

Pyrimidine Dimers ◽

Bulky Dna Lesions

In human cells, the oxidative DNA lesion 8,5′-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5′) next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5′-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

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Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

Molecular and Cellular Biology ◽

10.1128/mcb.26.3.777-788.2006 ◽

2006 ◽

Vol 26 (3) ◽

pp. 777-788 ◽

Cited By ~ 39

Author(s):

Yi Pei ◽

Hongyan Du ◽

Juliet Singer ◽

Courtney St. Amour ◽

Selena Granitto ◽

...

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Growth Defect ◽

Transcriptional Elongation ◽

Cyclin Dependent Kinase ◽

Elongation Complex ◽

Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

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Structural basis of INTAC-regulated transcription

10.1101/2021.11.29.470345 ◽

2021 ◽

Author(s):

Hai Zheng ◽

Qianwei Jin ◽

Yilun Qi ◽

Weida Liu ◽

Yulei Ren ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Biochemical Analysis ◽

Transcription Termination ◽

Structural Basis ◽

Transcription Start ◽

Elongation Complex ◽

Pol Ii ◽

Multiple Contacts ◽

Eukaryotic Genes ◽

Productive Elongation

For the majority of expressed eukaryotic genes, RNA polymerase II (Pol II) forms a paused elongation complex (PEC) and undergoes promoter-proximal pausing downstream of the transcription start site. The polymerase either proceeds into productive elongation or undergoes promoter-proximal premature transcription termination. It remains incompletely understood how transcription is regulated at this stage. Here, we determined the structure of PEC bound to INTAC, an Integrator-containing PP2A complex, at near-atomic resolution. The structure shows that INTAC partially wraps around PEC through multiple contacts, permitting the memetic nascent RNA to run into substrate-entry tunnel of the endonuclease subunit INTS11 of INTAC for cleavage. Pol II C-terminal domain (CTD) winds over INTAC backbone module through multiple anchors and is suspended above the phosphatase of INTAC for dephosphorylation. Biochemical analysis shows that INTAC-PEC association requires unphosphorylated CTD and could tolerate CTD phosphorylation, suggesting an INTAC-mediated persistent CTD dephosphorylation followed by reinforcement of the INTAC-PEC complex. Our study reveals how INTAC binds PEC and orchestrates RNA cleavage and CTD dephosphorylation, two critical events in generating premature transcription termination.

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RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2114065119 ◽

2022 ◽

Vol 119 (3) ◽

pp. e2114065119

Author(s):

Juntaek Oh ◽

Tiezheng Jia ◽

Jun Xu ◽

Jenny Chong ◽

Peter B. Dervan ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Minor Groove ◽

Dna Lesions ◽

Structural Basis ◽

Rna Pol Ii ◽

Pol Ii ◽

X Ray ◽

A Minor ◽

Insight Into

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

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Structure of the p53/RNA polymerase II assembly

Communications Biology ◽

10.1038/s42003-021-01934-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Shu-Hao Liou ◽

Sameer K. Singh ◽

Robert H. Singer ◽

Robert A. Coleman ◽

Wei-Li Liu

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Conformational Changes ◽

P53 Protein ◽

Binding Activity ◽

Transactivation Domain ◽

Pol Ii ◽

Dna Binding Activity ◽

Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

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DIPG-03. THERAPEUTIC TARGETING OF TRANSCRIPTIONAL ELONGATION IN DIFFUSE INTRINSIC PONTINE GLIOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa222.055 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii287-iii287

Author(s):

Hiroaki Katagi ◽

Nozomu Takata ◽

Yuki Aoi ◽

Yongzhan Zhang ◽

Emily J Rendleman ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Xenograft Model ◽

Diffuse Intrinsic Pontine Glioma ◽

Transcriptional Elongation ◽

Pontine Glioma ◽

Elongation Complex ◽

Pol Ii ◽

Repair Genes ◽

Novel Therapeutic

Abstract Diffuse intrinsic pontine glioma (DIPG) is highly aggressive brain stem tumor and needed to develop novel therapeutic agents for the treatment. The super elongation complex (SEC) is essential for transcription elongation through release of RNA polymerase II (Pol II). We found that AFF4, a scaffold protein of the SEC, is required for the growth of H3K27M-mutant DIPG cells. In addition, the small molecule SEC inhibitor, KL-1, increased promoter-proximal pausing of Pol II, and reduced transcription elongation, resulting in down-regulate cell cycle, transcription and DNA repair genes. KL-1 treatment decreased cell growth and increased apoptosis in H3K27M-mutant DIPG cells, and prolonged animal survival in our human H3K27M-mutant DIPG xenograft model. Our results demonstrate that the SEC disruption by KL-1 is a novel therapeutic strategy for H3K27M-mutant DIPG.

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The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat

eLife ◽

10.7554/elife.00327 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 43

Author(s):

Ursula Schulze-Gahmen ◽

Heather Upton ◽

Andrew Birnberg ◽

Katherine Bao ◽

Seemay Chou ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Elongation Factor ◽

Regulatory Proteins ◽

Tat Protein ◽

Elongation Complex ◽

Cyclin T1 ◽

Subunit Interface ◽

Hiv Tat ◽

Gene Transcripts ◽

Hiv 1

Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS.

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Structural basis of TFIIH activation for nucleotide excision repair

10.1101/628032 ◽

2019 ◽

Author(s):

Goran Kokic ◽

Aleksandar Chernev ◽

Dimitry Tegunov ◽

Christian Dienemann ◽

Henning Urlaub ◽

...

Keyword(s):

Dna Repair ◽

Transcription Factor ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Lesions ◽

Structural Basis ◽

Disease Mutations ◽

Mechanistic Analysis ◽

Nucleotide Excision ◽

Dna Complex

AbstractGenomes are constantly threatened by DNA damage, but cells can remove a large variety of DNA lesions by nucleotide excision repair (NER)1. Mutations in NER factors compromise cellular fitness and cause human diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome and trichothiodystrophy2,3. The NER machinery is built around the multisubunit transcription factor IIH (TFIIH), which opens the DNA repair bubble, scans for the lesion, and coordinates excision of the damaged DNA single strand fragment1,4. TFIIH consists of a kinase module and a core module that contains the ATPases XPB and XPD5. Here we prepare recombinant human TFIIH and show that XPB and XPD are stimulated by the additional NER factors XPA and XPG, respectively. We then determine the cryo-electron microscopy structure of the human core TFIIH-XPA-DNA complex at 3.6 Å resolution. The structure represents the lesion-scanning intermediate on the NER pathway and rationalizes the distinct phenotypes of disease mutations. It reveals that XPB and XPD bind double- and single-stranded DNA, respectively, consistent with their translocase and helicase activities. XPA forms a bridge between XPB and XPD, and retains the DNA at the 5’-edge of the repair bubble. Biochemical data and comparisons with prior structures6,7 explain how XPA and XPG can switch TFIIH from a transcription factor to a DNA repair factor. During transcription, the kinase module inhibits the repair helicase XPD8. For DNA repair, XPA dramatically rearranges the core TFIIH structure, which reorients the ATPases, releases the kinase module and displaces a ‘plug’ element from the DNA-binding pore in XPD. This enables XPD to move by ~80 Å, engage with DNA, and scan for the lesion in a XPG-stimulated manner. Our results provide the basis for a detailed mechanistic analysis of the NER mechanism.

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