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Towards a better understanding of DNA repair and related diseases

The TTDA protein is fused to the protein LacR and to a fluorescent protein (GFP) in order to form a chimeric protein, able to recognize LacO sequences (green spots). When TTDA is mutated (right), the protein is fixed on the genome (top right) but XPA (red) is not recruited downstream (no spot in the bottom right).

Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin.

Ziani S(1), Nagy Z(1), Alekseev S(1), Soutoglou E(1), Egly JM(1), Coin F(2).

J Cell Biol Sept. 1, 2014


Aug. 25, 2014

The research team of Frédéric Coin has just highlighted new mechanisms of the nucleotide excision DNA repair (NER) pathway, allowing them to take a new step in understanding trichothyodystrophy, a genetic disease that alters this process. Their findings are published on August 25th in the Journal of Cell Biology.


The DNA repair in question
The NER repair pathway allows the organism to detect specific lesions in the genome, to open the DNA molecule on the affected area, to remove the lesion and then to replace it with a repaired DNA sequence. All these steps involve more than 20 polypeptides which arrange themselves in a specific sequential order: XPC then TFIIH (containing XPB, XPD and TTDA) and XPA, etc. Researchers have tried to force the formation of the NER complex on DNA in the absence of damage, in order to observe whether the process could be conducted on a non-damaged DNA.

 

Forcing the recruitment of NER precursors onto DNA
The Lactose Operator-Lactose Repressor system (LacO-LacR), based on an endogenous existing system of transcription repression in bacteria, can force the binding of proteins of interest on the DNA without the presence of sequences or structures representing their natural targets. Researchers have fused the LacR protein to their protein of interest and expressed the chimera in a mammalian cells, in which the LacO sequence has been artificially introduced into the genome. The chimera is then recognizing the LacO sequence due to the presence of LacR. The researchers can then study the formation of protein complexes on the LacO by using immunofluorescence techniques.
Here the researchers forced NER proteins to bind to DNA in the absence of lesions, including the XPC protein which is responsible for the detection of DNA damage in the NER .To their surprise, they found that the steps of recruitment of the different proteins for the establishment of NER mechanism remained unchanged and were held in an orderly manner, in the same way as when these proteins recognize damaged DNA in a natural context.

 

Better understand trichothyodystrophia
The researchers also used the LacO-LacR system to highlight the molecular defects associated with a rare disease. Trichothyodystrophy is one of the genetic diseases that specifically affect the DNA repair mechanism. One of the most common mutations of the disease concerns TTDA, a subunit of the TFIIH complex opening the DNA molecule around the lesion. The mechanisms underlying this mutation were still unknown. Indeed, while it decreased the amount of TFIIH by 50%, only 10 to 20% of the effectiveness of the repair was maintained, suggesting other downstream effects. Here the researchers used the LacO-LacR system to fix the TTDA protein, mutated or not, and observe the impact on subsequent recruitment of repair proteins. They could demonstrat an alteration in the recruitment of the XPA protein, that would explain the significant decrease in the efficiency of DNA repair in patients with TTD-A.

 

These results show that the protein assembly of NER is independent of the presence of a lesion. They also bring new elements to the understanding of the NER mechanisms, and in particular in the case of the TTDA mutation responsible for trichothyodystrophy. This methodology can now be extended to other mutations responsible for alteration of the DNA repair mechanism and has great potential to better understand several rare genetic diseases.

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