The interaction of HELB with RPA and its role in human fertility

The overarching objective of this work is to improve our understanding of the molecular basis for human fertility. It has recently become apparent that there are strong links between proteins which replicate and repair DNA molecules and fertility traits including age at menopause and ovarian ageing. This relationship is thought to reflect the important role played by the DNA repair machinery in the generation of sperm and egg cells and the maintenance of the hereditary information they contain.

This project focusses on one particular DNA replication and repair factor called DNA helicase B (HELB). Geneticists have shown that mutation of this protein is strongly implicated in early onset menopause but we have no information at all about how these molecular defects in HELB affect its cellular functions. This partly reflects the fact that HELB is a poorly-studied protein; although several studies have shown that HELB contributes to DNA maintenance its precise roles in these pathways remain undefined. Furthermore, we have no understanding of the architecture of the HELB protein, nor of the complexes it forms with other replication and repair factors. We recently found one important clue to the function of HELB when we showed that it acts as a DNA motor protein to strip RPA from single-stranded DNA (ssDNA). RPA is a protein which binds rapidly and tightly to ssDNA to protect it from degradation, and the RPA-ssDNA filaments that are formed are key intermediates in many replication and repair processes. However, the subsequent removal of RPA from these intermediates is essential for the binding of downstream factors which complete replication and repair. Consequently, we have suggested that RPA remodeling is the underlying role of HELB in all of the varied pathways it has been associated with. Remarkably, structural modelling studies now suggest that the HELB defects associated with early onset menopause affect its ability to interact with RPA and this hypothesis is the basis for our research proposal.

In this work we will:

(1) Determine the architecture of the HELB-RPA complex to unveil the atomic details of the interface formed between HELB and RPA. In this way we will test how defects in HELB affect the binding of HELB to RPA.

(2) Determine the importance of the HELB-RPA interface, and of the HELB mutations that we think disrupt it, for HELB activity on RPA filaments and HELB-dependent DNA repair.

Our project will generate novel insights into the structure and function of HELB at the molecular level, and also the links between genetic defects in HELB, structural perturbations of the HELB-RPA interface, dysfunctional RPA filament remodeling, genetic instability and (ultimately) human fertility problems. This basic research will improve our broader understanding of human reproductive performance and will support further translational research to address infertility.

In accordance with an emerging role for DNA replication and repair factors in human fertility traits, the human DNA helicase B (HELB) has been implicated in menopause timing by GWAS studies. HELB is a poorly-characterized protein whose cellular roles are unclear. It has been reported to function in DNA replication initiation, the recovery from replication stress and the recombinational repair of DNA breaks. All of these pathways share RPA-ssDNA filaments as a common intermediate species.

We have recently shown that HELB is a DNA motor protein which is specifically recruited to RPA-ssDNA filaments and catalyses the processive removal of the tightly-bound RPA to leave naked ssDNA. We postulated that this facilitates the hand-off of ssDNA intermediates to downstream replication and repair factors. Intriguingly, modelling suggests that variants of HELB implicated in early onset menopause contain missense mutations which map to the HELB-RPA interface. However, no structural information is currently available for HELB and the complexes it forms, and so the links between HELB structural perturbation, defective HELB function and fertility remain unexplored.

In this project we will study the architecture of the HELB-RPA-ssDNA complex and how this might be affected in the fertility variants using an integrated structural biology approach combining modelling, cryo-EM, native- and HDX-MS, and biophysical analysis of mutant proteins. This will facilitate the generation of designed mutant proteins and nanobodies targeting different aspects of HELB function including its interaction with RPA. These tools will then be used to probe HELB function, the role of the RPA interface, and the effects of fertility variants using in vitro activity assays and cell-based DNA repair assays.