Researchers from the Francis Crick Institute, the MRC London Institute of Medical Sciences (LMS) and the Memorial Sloan Kettering Cancer Center (MSKCC) have shown for the first time that an enzyme called HELQ repairs DNA double-stranded breaks via two distinct activities that are enhanced through its interactions with two specific DNA repair proteins.

DNA double-stranded breaks (DSBs) are relatively rare, potentially devastating occurrences for a cell. Their incorrect repair has been linked to cancer and infertility. The Crick’s Boulton laboratory previously showed that HELQ, a member of the superfamily-2 of helicase enzymes, prevents germ cell attrition and tumorigenesis, but exactly how it works in DNA repair had alluded scientists for years.

Through the use of biochemistry analysis and single-molecular imaging, Bolton’s lab, the LMS Single Molecule Imaging Research Group and the MSKCC Department of Oncology have shown HELQ can mend DSBs by virtue of two distinct activities that are enhanced through its interaction with two proteins, RAD51 and RPA.

In human cells, loss of HELQ was previously shown to compromise DNA repair leading to persistence of the DSB repair protein RAD51. This raised the possibility that RAD51 and HELQ could be working together during DSB repair.

To unpick the potential co-activities of RAD51 and HELQ, Crick and LMS researchers utilised an optical tweezer set-up combined with microfluidics and confocal microscopy (C-TRAP). This enables single molecule imagery, meaning they can focus on proteins interacting on a single piece of DNA. They fluorescently labelled RAD51 so they could track its movements during DSB repair. In the absence of HELQ, they found RAD51 binds to regions of DNA damage, but remains static. However, upon addition of HELQ, RAD51 was found to bind to HELQ and it remained bound as HELQ moved along the damaged DNA unwinding it as it moved. Interestingly, HELQ was able to unwind DNA in the absence of RAD51, but it was much faster when RAD51 was present. In fact, the data suggests RAD51 and HELQ form a complex that unwinds DNA at a rate of approximately threefold faster compared with HELQ alone.

Professor David Rueda, Head of the LMS Single Molecule Imaging Group said: “Our genetic material, our DNA, is under constant attack and suffers tens of thousands of daily chemical damages. If left unrepaired, these damages can cause serious diseases such as cancer. Our cells have evolved elegant systems to repair damaged DNA. Numerous proteins are involved in repair pathways. Teasing out the function of each protein involved in these pathways has been a challenging scientific question for decades. Single-molecule imaging is a powerful way to address such questions because it enables us to see and manipulate the function of each protein in these repair pathways. Specifically, here we have been able to show that HELQ functions as a swiss-knife protein with multiple functions adapted for more than one repair pathway. Such findings will pave the way for future cancer therapies.”

RPA is known to bind to single-stranded DNA and to stop it from curling in on itself, keeping the DNA in an optimal position and shape for its repair. RPA has been shown to stop HELQ-induced unwinding of DNA, suggesting that there could some interaction between the two proteins.

LMS, Crick and MSKCC researchers found that while HELQ does initially unwind the DNA for repair, it also assists in the alignment and annealing of complementary single-strands so that they can reform a double-stranded piece of DNA. HELQ can promote this DNA reannealing activity alone, but interestingly, addition of RPA could stimulate HELQ-DNA annealing activity by around two-fold. This suggests RPA inhibits DNA unwinding by HELQ but strongly stimulates DNA strand annealing.

Professor Simon Boulton, Head of the Crick’s DSB Repair Metabolism Laboratory said: “This work is the culmination of nearly a decade’s work trying to understand how HELQ functions in DNA repair. The application of biophysical approaches permitting the direct visualisation of single molecules of HELQ acting on DNA, coupled with cellular studies, have revealed HELQ as an exquisitely regulated DNA unwinding enzyme that also possesses a novel annealing function. When combined these activities are key to facilitate DSB repair. These insights help us explain why HELQ loss confers genome instability, infertility and cancer predisposition in mammals.”

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51 was published in Nature on 22 December 2021.