scholarly journals The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

2019 ◽  
Author(s):  
JT Barnett ◽  
J Kuper ◽  
W Koelmel ◽  
C Kisker ◽  
NM Kad
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Binding ◽  
Nucleotide Excision Repair ◽  
Single Molecule ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
The Core ◽  
Nucleotide Excision

AbstractNucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in mammalian NER genes lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and five ‘structural’ subunits. Two of these structural TFIIH proteins, p44 and p62 remain relatively unstudied; p44 is known to regulate the helicase activity of XPD during NER whereas p62’s role is thought to be structural. However, a recent cryo-EM structure shows that p44, p62, and XPD make extensive contacts within TFIIH, with part of p62 occupying XPD’s DNA binding site. This observation implies a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPD’s ATPase but upon encountering DNA damage, further stimulation is only observed when p62 is part of the ternary complex; suggesting a role for the p44/p62 heterodimer in TFIIH’s mechanism of damage detection. Using single molecule imaging, we demonstrate that p44/p62 independently interacts with DNA; it is seen to diffuse, however, in the presence of UV-induced DNA lesions the complex stalls. Combined with the analysis of a recent cryo-EM structure we suggest that p44/p62 acts as a novel DNA-binding entity within TFIIH that is capable of recognizing DNA damage. This revises our understanding of TFIIH and prompts more extensive investigation into the core subunits for an active role during both DNA repair and transcription.

scholarly journals The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

2020 ◽  
Vol 48 (22) ◽  
pp. 12689-12696
Author(s):  
Jamie T Barnett ◽  
Jochen Kuper ◽  
Wolfgang Koelmel ◽  
Caroline Kisker ◽  
Neil M Kad
Keyword(s):  
Dna Binding ◽  
Nucleotide Excision Repair ◽  
Single Molecule ◽  
Excision Repair ◽  
Active Role ◽  
Dna Lesions ◽  
Binding Studies ◽  
The Core ◽  
Nucleotide Excision ◽  
Direct Binding

Abstract Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five ‘structural’ subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD’s DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD’s affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62’s high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.

scholarly journals DICER- and MMSET-catalyzed H4K20me2 recruits the nucleotide excision repair factor XPA to DNA damage sites

2017 ◽  
Vol 217 (2) ◽  
pp. 527-540 ◽  
Author(s):  
Shalaka Chitale ◽  
Holger Richly
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Histone H4 ◽  
Damage Site ◽  
Nucleotide Excision ◽  
Lesion Recognition

Ultraviolet (UV) irradiation triggers the recruitment of DNA repair factors to the lesion sites and the deposition of histone marks as part of the DNA damage response. The major DNA repair pathway removing DNA lesions caused by exposure to UV light is nucleotide excision repair (NER). We have previously demonstrated that the endoribonuclease DICER facilitates chromatin decondensation during lesion recognition in the global-genomic branch of NER. Here, we report that DICER mediates the recruitment of the methyltransferase MMSET to the DNA damage site. We show that MMSET is required for efficient NER and that it catalyzes the dimethylation of histone H4 at lysine 20 (H4K20me2). H4K20me2 at DNA damage sites facilitates the recruitment of the NER factor XPA. Our work thus provides evidence for an H4K20me2-dependent mechanism of XPA recruitment during lesion recognition in the global-genomic branch of NER.

scholarly journals Recognition of DNA damage by XPC coincides with disruption of the XPC–RAD23 complex

2012 ◽  
Vol 196 (6) ◽  
pp. 681-688 ◽  
Author(s):  
Steven Bergink ◽  
Wendy Toussaint ◽  
Martijn S. Luijsterburg ◽  
Christoffel Dinant ◽  
Sergey Alekseev ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Deoxyribonucleic Acid ◽  
Excision Repair ◽  
Proteasomal Degradation ◽  
Repair Process ◽  
Dna Lesions ◽  
Direct Role ◽  
Nucleotide Excision

The recognition of helix-distorting deoxyribonucleic acid (DNA) lesions by the global genome nucleotide excision repair subpathway is performed by the XPC–RAD23–CEN2 complex. Although it has been established that Rad23 homologs are essential to protect XPC from proteasomal degradation, it is unclear whether RAD23 proteins have a direct role in the recognition of DNA damage. In this paper, we show that the association of XPC with ultraviolet-induced lesions was impaired in the absence of RAD23 proteins. Furthermore, we show that RAD23 proteins rapidly dissociated from XPC upon binding to damaged DNA. Our data suggest that RAD23 proteins facilitate lesion recognition by XPC but do not participate in the downstream DNA repair process.

scholarly journals Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex

2016 ◽  
Vol 113 (16) ◽  
pp. E2296-E2305 ◽  
Author(s):  
Yogambigai Velmurugu ◽  
Xuejing Chen ◽  
Phillip Slogoff Sevilla ◽  
Jung-Hyun Min ◽  
Anjum Ansari
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Nucleotide Excision Repair ◽  
Base Pair ◽  
Binding Proteins ◽  
Excision Repair ◽  
Conformational Dynamics ◽  
Dna Binding Proteins ◽  
Dna Lesions ◽  
Nucleotide Excision

DNA damage repair starts with the recognition of damaged sites from predominantly normal DNA. In eukaryotes, diverse DNA lesions from environmental sources are recognized by the xeroderma pigmentosum C (XPC) nucleotide excision repair complex. Studies of Rad4 (radiation-sensitive 4; yeast XPC ortholog) showed that Rad4 “opens” up damaged DNA by inserting a β-hairpin into the duplex and flipping out two damage-containing nucleotide pairs. However, this DNA lesion “opening” is slow (˜5–10 ms) compared with typical submillisecond residence times per base pair site reported for various DNA-binding proteins during 1D diffusion on DNA. To address the mystery as to how Rad4 pauses to recognize lesions during diffusional search, we examine conformational dynamics along the lesion recognition trajectory using temperature-jump spectroscopy. Besides identifying the ˜10-ms step as the rate-limiting bottleneck towards opening specific DNA site, we uncover an earlier ˜100- to 500-μs step that we assign to nonspecific deformation (unwinding/“twisting”) of DNA by Rad4. The β-hairpin is not required to unwind or to overcome the bottleneck but is essential for full nucleotide-flipping. We propose that Rad4 recognizes lesions in a step-wise “twist-open” mechanism, in which preliminary twisting represents Rad4 interconverting between search and interrogation modes. Through such conformational switches compatible with rapid diffusion on DNA, Rad4 may stall preferentially at a lesion site, offering time to open DNA. This study represents the first direct observation, to our knowledge, of dynamical DNA distortions during search/interrogation beyond base pair breathing. Submillisecond interrogation with preferential stalling at cognate sites may be common to various DNA-binding proteins.

scholarly journals Structural basis of TFIIH activation for nucleotide excision repair

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.

scholarly journals Databases and Bioinformatics Tools for the Study of DNA Repair

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Kaja Milanowska ◽  
Kristian Rother ◽  
Janusz M. Bujnicki
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Excision Repair ◽  
Uv Light ◽  
Dna Lesions ◽  
Environmental Chemicals ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Repair Mechanisms ◽  
Base Excision

DNA is continuously exposed to many different damaging agents such as environmental chemicals, UV light, ionizing radiation, and reactive cellular metabolites. DNA lesions can result in different phenotypical consequences ranging from a number of diseases, including cancer, to cellular malfunction, cell death, or aging. To counteract the deleterious effects of DNA damage, cells have developed various repair systems, including biochemical pathways responsible for the removal of single-strand lesions such as base excision repair (BER) and nucleotide excision repair (NER) or specialized polymerases temporarily taking over lesion-arrested DNA polymerases during the S phase in translesion synthesis (TLS). There are also other mechanisms of DNA repair such as homologous recombination repair (HRR), nonhomologous end-joining repair (NHEJ), or DNA damage response system (DDR). This paper reviews bioinformatics resources specialized in disseminating information about DNA repair pathways, proteins involved in repair mechanisms, damaging agents, and DNA lesions.

scholarly journals Can vitamin C induce nucleotide excision repair? Support from in vitro evidence

2009 ◽  
Vol 103 (5) ◽  
pp. 686-695 ◽  
Author(s):  
Ruth J. Bevan ◽  
Nalini Mistry ◽  
Parul R. Patel ◽  
Eugene P. Halligan ◽  
Rosamund Dove ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Stress Response ◽  
Vitamin C ◽  
Nucleotide Excision Repair ◽  
Mononuclear Cells ◽  
Culture Supernatant ◽  
Excision Repair ◽  
Peripheral Blood Mononuclear ◽  
Nucleotide Excision

Intracellular vitamin C acts to protect cells against oxidative stress by intercepting reactive oxygen species (ROS) and minimising DNA damage. However, rapid increases in intracellular vitamin C may induce ROS with subsequent DNA damage priming DNA repair processes. Herein, we examine the potential of vitamin C and the derivative ascorbate-2-phosphate (2-AP) to induce a nucleotide excision repair (NER) response to DNA damage in a model of peripheral blood mononuclear cells. Exposure of cells to elevated levels of vitamin C induced ROS activity, resulting in increased levels of deoxycytidine glyoxal (gdC) and 8-oxo-2′-deoxyguanosine (8-oxodG) adducts in DNA; a stress response was also induced by 2-AP, but was delayed in comparison to vitamin C. Evidence of gdC repair was also apparent. Measurement of cyclobutane thymine–thymine dimers (T < >T) in DNA and culture supernatant were included as a positive marker for NER activity; this was evidenced by a reduction in DNA and increases in culture supernatant levels of T < >T for vitamin C-treated cells. Genomics analysis fully supported these findings confirming that 2-AP, in particular, induced genes associated with stress response, cell cycle arrest, DNA repair and apoptosis, and additionally provided evidence for the involvement of vitamin C in the mobilisation of intracellular catalytic Fe.

scholarly journals Nucleotide Excision Repair and Transcription-coupled DNA Repair Abrogate the Impact of DNA Damage on Transcription

2015 ◽  
Vol 291 (2) ◽  
pp. 848-861 ◽  
Author(s):  
Aditi Nadkarni ◽  
John A. Burns ◽  
Alberto Gandolfi ◽  
Moinuddin A. Chowdhury ◽  
Laura Cartularo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Nucleotide Excision ◽  
The Impact

scholarly journals Mechanism of DNA Lesion Homing and Recognition by the Uvr Nucleotide Excision Repair System

Research ◽  
2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Seung-Joo Lee ◽  
Rou-Jia Sung ◽  
Gregory L. Verdine
Keyword(s):  
Dna Repair ◽  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Dna Lesions ◽  
Repair System ◽  
Genome Maintenance ◽  
Dna Lesion ◽  
Nucleotide Excision ◽  
Biochemical Studies ◽  
Lesion Recognition

Nucleotide excision repair (NER) is an essential DNA repair system distinguished from other such systems by its extraordinary versatility. NER removes a wide variety of structurally dissimilar lesions having only their bulkiness in common. NER can also repair several less bulky nucleobase lesions, such as 8-oxoguanine. Thus, how a single DNA repair system distinguishes such a diverse array of structurally divergent lesions from undamaged DNA has been one of the great unsolved mysteries in the field of genome maintenance. Here we employ a synthetic crystallography approach to obtain crystal structures of the pivotal NER enzyme UvrB in complex with duplex DNA, trapped at the stage of lesion-recognition. These structures coupled with biochemical studies suggest that UvrB integrates the ATPase-dependent helicase/translocase and lesion-recognition activities. Our work also conclusively establishes the identity of the lesion-containing strand and provides a compelling insight to how UvrB recognizes a diverse array of DNA lesions.

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