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Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways - PubMed

  • ️Sun Jan 01 2017

Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways

Masahiko Okuda et al. Nucleic Acids Res. 2017.

Abstract

Nucleotide excision repair is initiated by two different damage recognition subpathways, global genome repair (GGR) and transcription-coupled repair (TCR). In GGR, XPC detects DNA lesions and recruits TFIIH via interaction with the pleckstrin homology (PH) domain of TFIIH subunit p62. In TCR, an elongating form of RNA Polymerase II detects a lesion on the transcribed strand and recruits TFIIH by an unknown mechanism. Here, we found that the TCR initiation factor UVSSA forms a stable complex with the PH domain of p62 via a short acidic string in the central region of UVSSA, and determined the complex structure by NMR. The acidic string of UVSSA binds strongly to the basic groove of the PH domain by inserting Phe408 and Val411 into two pockets, highly resembling the interaction mechanism of XPC with p62. Mutational binding analysis validated the structure and identified residues crucial for binding. TCR activity was markedly diminished in UVSSA-deficient cells expressing UVSSA mutated at Phe408 or Val411. Thus, a common TFIIH recruitment mechanism is shared by UVSSA in TCR and XPC in GGR.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

Amino acid sequence and domain organization of UVSSA. (A) Alignment of the amino acid sequences of UVSSA and the TFIIH p62 PH-binding regions of human XPC, TFIIEα, p53, and DP1. Green and orange circles above the sequence of XPC indicate the amino acids that insert into pocket 1 and pocket 2 in p62 PH, respectively. (B) Domain organization of TFIIH p62 and UVSSA. Intrinsically disordered regions in UVSSA predicted by IDEAL (55) are indicated by the black bars.

Figure 2.
Figure 2.

Specific binding of UVSSA to TFIIH p62 PH. (A) Isothermal titration calorimetry for the binding of UVSSA390−434 to TFIIH p62 PH. Shown are thermogram (upper panel) and binding isotherm (lower panel) of the calorimetric titration. Titrant: wild-type UVSSA390–434. (B) NMR chemical shift perturbation for 15N-labeled UVSSA390−434 with TFIIH p62 PH (upper panel) and for 15N-labeled TFIIH p62 PH with UVSSA390−434 (lower panel). Shown in overlay of 1H–15N HSQC spectra before (black) and after (blue) the addition of unlabeled sample. Selected regions are shown here (see full region in Supplementary Figures S1 and S2). Residues showing a large chemical shift change are labeled.

Figure 3.
Figure 3.

Structure of the complex between UVSSA and TFIIH p62 PH. (A) The 20 best solution structures represented as a line diagram. UVSSA and p62 are shown in magenta and black, respectively. (B–E) The 20 best solution structures represented as a ribbon diagram. UVSSA and p62 are shown in magenta and blue, respectively. For clarity, the four N-terminal residues and the nine C-terminal residues of UVSSA390–434 have been omitted. The structures in C, D, and E are rotated by 45, -45, and 180 degrees, respectively, relative to the structures in A and B.

Figure 4.
Figure 4.

Structural comparison of the UVSSA–p62 PH and XPC–p62 PH complexes. (A) Superimposition of the best 20 structures of the UVSSA–p62 PH complex (magenta/pink) and those of the XPC–p62 PH complex (green/grey). (B) Binding surface of p62 PH in complex with UVSSA (left) and XPC (right). The electrostatic potential surfaces are shown. Positive potential is shown in blue, and negative potential in red. UVSSA and XPC are shown in stick representation (best 20 structures).

Figure 5.
Figure 5.

Comparison of p62 PH recognition between TCR factor UVSSA and GGR factor XPC. (A, C, E, G) Intermolecular interactions in the UVSSA–p62 PH complex. (B, D, F, H) Intermolecular interactions in the XPC–p62 PH complex. p62, UVSSA, and XPC are shown in blue, magenta, and green, respectively. To discriminate p62 from UVSSA or XPC, residues of p62 are labeled in lowercase and residues of UVSSA or XPC are labeled in uppercase.

Figure 6.
Figure 6.

ITC analysis of p62 PH binding by UVSSA mutant. Shown are thermograms (upper panels) and binding isotherms (lower panels) of the calorimetric titration. (A) Titrant: wild type UVSSA399–419. (B) Titrant: F408A UVSSA399–419. (C) Titrant: V411A UVSSA399–419. (D) Titrant: V409A UVSSA399–419. The calculated binding dissociation constant (Kd) is shown below each panel. Data are the mean ± standard error (S.D.) from the fitting curve.

Figure 7.
Figure 7.

The p62 binding domain of UVSSA is essential for transcription coupled NER activity. (A) Recovery of RNA synthesis was measured in Kps3 cells expressing wild-type UVSSA–V5 or UVSSA–V5 with mutations in the p62-binding domain (filled bars, 12 J/m2 of UV-C; open bars, no UV). Aromatic (green) and hydrophobic (orange) residues required for strong binding between UVSSA and p62 are indicated. Error bars represent the standard error (S.E.) of quadrupled experiments. (B) The interaction of p62 with either wild-type UVSSA or various UVSSA mutant proteins was assayed by immunoprecipitation, followed by western blotting. CL, crude lysate; IP, immunoprecipitate.

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References

    1. Lehmann A.R. DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie. 2003; 85:1101–1111. - PubMed
    1. Shuck S.C., Short E.A., Turchi J.J.. Eukaryotic nucleotide excision repair: from understanding mechanisms to influencing biology. Cell Res. 2008; 18:64–72. - PMC - PubMed
    1. Naegeli H., Sugasawa K.. The xeroderma pigmentosum pathway: decision tree analysis of DNA quality. DNA Repair. 2011; 10:673–683. - PubMed
    1. Fousteri M., Mullenders L.H.. Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects. Cell Res. 2008; 18:73–84. - PubMed
    1. Hanawalt P.C., Spivak G.. Transcription-coupled DNA repair: two decades of progress and surprises. Nat. Rev. Mol. Cell Biol. 2008; 9:958–970. - PubMed

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