Probing the function of the fertility-related HELB protein in DNA repair

Abstract

Background: The timing of menopause has major health consequences, with both early and late menopause associated with increased disease risk [1-3]. Genetic studies increasingly implicate DNA damage response (DDR) factors in determining age at natural menopause (ANM) [4-7]. Among these, Human DNA helicase B (HELB) is a strong candidate regulator [4, 8, 9]. HELB functions in DNA replication and repair, but its role in homologous recombination (HR) remains disputed, with evidence for it both promoting and inhibiting this pathway [9-11]. Clarifying this role is important for understanding oocyte genome stability and reproductive lifespan.
Methods: We investigated HELB function in HR and single strand annealing (SSA) using reporter assays and orthogonal protein depletion strategies. HR and SSA were assessed using the DR-GFP and SA-GFP reporters in U2OS cells as well as DSB-Spectrum V1/V3 reporters in HEK293T cells. HELB was depleted with siRNAs and matched controls from three different companies (Santa Cruz, Dharmacon, and Invitrogen Silencer Select) and knockout clones were generated by CRISPR-Cas9.
Results: All HELB-targeting siRNAs reduced HELB protein expression, but effects on DNA repair varied by reagent and reporter. Silencer HELB siRNA consistently decreased HR and SSA, reaching significance in V1, V3 and SA-GFP. Santa Cruz HELB siRNA significantly reduced SSA but had minimal effects in the HR reporters. Dharmacon HELB siRNA showed apparent increases in both HR and SSA, but this was attributed to off-target effects of the control siRNA. HELB knockout DR-GFP lines showed variable HR ranging from no change to significant reduction, while a validated knockout in the V3 reporter showed significantly reduced HR.
Conclusions: Across orthogonal reporter systems and HELB depletion strategies, our results broadly support the idea that HELB is a positive regulator of both HR and SSA, although the magnitude of the effect depends on context and reagent.

Date of Award	9 Dec 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Mark S Dillingham (Supervisor) & Oliver J Wilkinson (Supervisor)