Molecular-level DNA repair could pave the way for more effective cancer drugs

To reduce the biological consequences of DNA damage, all living organisms have evolved mechanisms to tolerate and repair DNA damage to ensure that genetic information is accurately inherited.

One of these DNA repair pathways is nucleotide excision repair (NER), used by mammals to remove bulky DNA lesions such as those formed by UV light, environmental mutagens, and some cancer chemotherapeutic adducts from DNA.

DNA-repairing mechanism

NER is highly regulated and occurs in four main steps: (1) DNA-damage recognition, (2) unwinding of the DNA strand by molecular motor TFIIH and excision of about 30 nucleotides by the nucleases XPG and XPF to remove the damage, (3) gap-filling DNA synthesis and (4) ligation of open DNA ends.

Though researchers knew the leading players in the NER process, how these steps are coordinated and regulated was not well understood.

Now an international team led by researchers at King Abdullah University of Science and Technology (KAUST) and the University of Texas MD Anderson Cancer Center have shown how the NER mechanism is controlled at the molecular level, which is potentially significant for cancer treatment.

"During radiation therapy, cancer cells are blasted with radiation to shrink tumors. In this situation, however, NER works against the treatment, trying to repair the damage and preventing cell death, which significantly reduces the effectiveness of the treatment," said Ph.D. student and the study's lead author Amer Bralić.

Designing NER inhibitors as effective drugs

A significant obstacle in designing inhibitors is the lack of basic knowledge about the NER mechanism. Professor Samir Hamdan's group at KAUST has uncovered how TFIIH uses XPG to stimulate its motor activity to locate damaged DNA. Once TFIIH locates the damage, it signals the XPG nuclease activity to excise it.

"The finding unravels a fundamental control mechanism in NER and argues for tackling the interaction between TFIIH and XPG as an effective drug target," says Prof. Samir Hamdan.

A better understanding of the mechanisms involved in DNA repair paves the way for developing NER inhibitors to improve the effectiveness of radiation therapy.

Mutation in proteins involved in the NER mechanism mediates more than ten clinical diseases. A mutation in one of these proteins may cause several diseases, and different combinations of protein mutations may cause one disease. "Our mechanistic findings provide new perspectives on linking molecular level information to disease states," explains Bralić.

"Through the KAUST Smart Health Initiative, we will work with clinicians in the Kingdom to study the clinical mutational landscape of NER proteins in patients," concludes Hamdan.

The discovery was selected as a "breakthrough article" by the journal Nucleic Acids Research.

Abstract:

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5′-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5′-ssDNA, ensuring the correct ssDNA bubble size before cleavage.