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Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

ABSTRACT: The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer region yield yeast with enhanced sensitivity to UV irradiation. Isothermal titration calorimetry studies demonstrate that two acidic segments of the Rad2 spacer bind to Tfb1PH with nanomolar affinity. Structure determination of a Rad2-Tfb1PH complex indicates that Rad2 binds to TFIIH using a similar motif as TFIIE? uses to bind TFIIH in the PIC. Together, these results provide a mechanistic bridge between the role of TFIIH in transcription and DNA repair.

SUBMITTER: Lafrance-Vanasse J

PROVIDER: S-EPMC3384317 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

Lafrance-Vanasse Julien J Arseneault Geneviève G Cappadocia Laurent L Chen Hung-Ta HT Legault Pascale P Omichinski James G JG

Nucleic acids research 20120228 12

The general transcription factor IIH (TFIIH) plays crucial roles in transcription as part of the pre-initiation complex (PIC) and in DNA repair as part of the nucleotide excision repair (NER) machinery. During NER, TFIIH recruits the 3'-endonuclease Rad2 to damaged DNA. In this manuscript, we functionally and structurally characterized the interaction between the Tfb1 subunit of TFIIH and Rad2. We show that deletion of either the PH domain of Tfb1 (Tfb1PH) or several segments of the Rad2 spacer ...[more]

PMID: 22373916

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Project description:Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of intrinsically disordered acidic TADs in recognizing common target host proteins.

Project description:The coagulation factor XIII(FXIII) is a plasma circulating heterotetrameric protransglutaminase that acts at the end of the coagulation cascade by covalently cross-linking preformed fibrin clots (to themselves and to fibrinolytic inhibitors) in order to stabilize them against fibrinolysis. It circulates in the plasma as a heterotetramer composed of two homomeric catalytic Factor XIIIA2 (FXIIIA2) and two homomeric protective/carrier Factor XIIIB2 subunit (FXIIIB2). Congenital deficiency of FXIII is of two types: severe homozygous/compound heterozygous FXIII deficiency which results in severe bleeding symptoms and mild heterozygous FXIII deficiency which is associated with mild bleeding (only upon trauma) or an asymptomatic phenotype. Defects in the F13B gene (Factor XIIIB subunit) occur more frequently in mild FXIII deficiency patients than in severe FXIII deficiency. We had recently reported secretion-related defects for seven previously reported F13B missense mutations. In the present study we further analyze the underlying molecular pathological mechanisms as well as the heterozygous expression phenotype for these mutations using a combination of in vitro heterologous expression (in HEK293T cells) and confocal microscopy. In combination with the in vitro work we have also performed an in silico solvated molecular dynamic simulation study on previously reported FXIIIB subunit sushi domain homology models in order to predict the putative structure-functional impact of these mutations. We were able to categorize the mutations into the following functional groups that: (1) affect antigenic stability as well as binding to FXIIIA subunit, that is, Cys5Arg, Cys316Phe, and Pro428Ser (2) affect binding to FXIIIA subunit with little or no influence on antigenic stability, that is, Ile81Asn and Val401Gln c) influence neither aspects and are most likely causality linked polymorphisms or functional polymorphisms, that is, Leu116Phe and Val217Ile. The Cys5Arg mutation was the only mutation to show a direct secretion-based defect since the mutated protein was observed to accumulate in the endoplasmic reticulum.

Dataset Information

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

Publications

Structural and functional characterization of interactions involving the Tfb1 subunit of TFIIH and the NER factor Rad2.

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