Sequence-dependent modulation of nucleotide excision repair: the efficiency of the incision reaction is inversely correlated with the stability of the pre-incision UvrB-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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Chromosomal model for analysis of a long CTG/CAG tract stability in wild-type Escherichia coli and its nucleotide excision repair mutants

Canadian Journal of Microbiology ◽

10.1139/w07-047 ◽

2007 ◽

Vol 53 (7) ◽

pp. 860-868 ◽

Cited By ~ 4

Author(s):

Sylwia T. Szwarocka ◽

Paweł Stączek ◽

Paweł Parniewski

Keyword(s):

Escherichia Coli ◽

Nucleotide Excision Repair ◽

Trinucleotide Repeat ◽

Excision Repair ◽

Repetitive Sequences ◽

Lambda Phage ◽

E Coli ◽

Repeat Sequences ◽

Nucleotide Excision ◽

The Stability

Many human hereditary neurological diseases, including fragile X syndrome, myotonic dystrophy, and Friedreich’s ataxia, are associated with expansions of the triplet repeat sequences (TRS) (CGG/CCG, CTG/CAG, and GAA/TTC) within or near specific genes. Mechanisms that mediate mutations of TRS include DNA replication, repair, and gene conversion and (or) recombination. The involvement of the repair systems in TRS instability was investigated in Escherichia coli on plasmid models, and the results showed that the deficiency of some nucleotide excision repair (NER) functions dramatically affects the stability of long CTG inserts. In such models in which there are tens or hundreds of plasmid molecules in each bacterial cell, repetitive sequences may interact between themselves and according to a recombination hypothesis, which may lead to expansions and deletions within such repeated tracts. Since one cannot control interaction between plasmids, it is also sometimes difficult to give precise interpretation of the results. Therefore, using modified lambda phage (λInCh), we have constructed a chromosomal model to study the instability of trinucleotide repeat sequences in E. coli. We have shown that the stability of (CTG/CAG)68 tracts in the bacterial chromosome is influenced by mutations in NER genes in E. coli. The absence of the uvrC or uvrD gene products greatly enhances the instability of the TRS in the chromosome, whereas the lack of the functional UvrA or UvrB proteins causes substantial stabilization of (CTG/CAG) tracts.

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Post-incision steps of nucleotide excision repair in Escherichia coli. Disassembly of the UvrBC-DNA complex by helicase II and DNA polymerase I.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)48352-4 ◽

1992 ◽

Vol 267 (2) ◽

pp. 780-788 ◽

Cited By ~ 3

Author(s):

D K Orren ◽

C P Selby ◽

J E Hearst ◽

A Sancar

Keyword(s):

Escherichia Coli ◽

Nucleotide Excision Repair ◽

Dna Polymerase ◽

Excision Repair ◽

Dna Polymerase I ◽

Nucleotide Excision ◽

Polymerase I ◽

Dna Complex

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C. elegans TFIIH subunit GTF-2H5/TTDA is a non-essential transcription factor indispensable for DNA repair

10.1101/2021.06.04.447037 ◽

2021 ◽

Author(s):

Karen L. Thijssen ◽

Melanie van der Woude ◽

Carlota Davo-Martinez ◽

Mariangela Sabatella ◽

Wim Vermeulen ◽

...

Keyword(s):

Nucleotide Excision Repair ◽

Transcription Initiation ◽

Potential Model ◽

Excision Repair ◽

C Elegans ◽

Nucleotide Excision ◽

Or Gene ◽

The Stability ◽

Essential Transcription Factor

The 10-subunit TFIIH complex is vital to both transcription initiation and nucleotide excision repair. Hereditary mutations in its smallest subunit, TTDA/GTF2H5, cause a photosensitive form of the rare developmental brittle hair disorder trichothiodystrophy (TTD). Some TTD features are thought to be caused by subtle transcription or gene expression defects. Strikingly, TTDA/GTF2H5 knockout mice are not viable, which makes it difficult to investigate how TTDA/GTF2H5 promotes transcription in vivo. Here, we show that deficiency of the C. elegans TTDA ortholog GTF-2H5 is, however, compatible with viability and growth, in contrast to depletion of other TFIIH subunits. We also show that GTF-2H5 promotes the stability of TFIIH in multiple tissues and is indispensable for nucleotide excision repair, in which it facilitates recruitment of the TFIIH complex to DNA damage. Strikingly, when transcription is challenged, gtf-2H5 embryos die due to the intrinsic TFIIH fragility in the absence of GTF-2H5. These results support the idea that TTDA/GTF2H5 mutations cause transcription impairment underlying trichothiodystrophy and establish C. elegans as potential model for studying the pathogenesis of this disease.

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Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress.

10.1101/2021.07.10.451908 ◽

2021 ◽

Author(s):

Julien Brochu ◽

Emilie Vlachos-Breton ◽

Marc Drolet

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Dna Amplification ◽

Loop Formation ◽

E Coli ◽

Nucleotide Excision ◽

Type Ia ◽

Rnase Hi ◽

The Stability ◽

Null Mutants

E. coli type 1A topoisomerases (topos), topo I (topA) and topo III (topB) have both relaxation and decatenation activities. B. subtilis and E. coli topA topB null cells can survive owing to DNA amplifications allowing overproduction of topo IV, the main cellular decatenase that can also relax supercoiling. We show that overproducing human topo IB, a relaxase but not a decatenase, can substitute for topo IV in allowing E. coli topA null but not topA topB null cells to survive. Deleting topB exacerbates phenotypes of topA null mutants including hypernegative supercoiling, R-loop formation, and RNase HI-sensitive replication, phenotypes that are not corrected by topo IV overproduction. These phenotypes lead to Ter DNA amplification causing a chromosome segregation defect that is corrected by topo IV overproduction. Furthermore, topA topB null mutants not overproducing topo IV acquire uvrB or uvrC mutations, revealing a nucleotide excision repair (NER)-dependent problem with replication fork progression. Thus, type IA topos maintain the stability of the genome by providing essential relaxase and decatenase activities to prevent and solve topological stress related to R-loops and NER. Moreover, excess R-loop formation is well tolerated in cells that have enough topoisomerase activity to support the subsequent replication-related topological stress.

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