Rare SNP in the HELB gene interferes with RPA interaction and cellular function of HELB

Area of Science:

• Genetics and Molecular Biology
• Cellular Biology
• Reproductive Biology

Background:

• The human helicase HELB plays critical roles in DNA replication, stress response, and DNA double-strand break repair.
• A single-nucleotide polymorphism (SNP), rs75770066, in the HELB gene has been associated with variations in the age at natural menopause (ANM).

Purpose of the Study:

• To investigate the functional impact of the rs75770066 polymorphism on HELB protein function and its cellular consequences.
• To elucidate the molecular mechanism by which the D506G substitution in HELB affects its interaction with RPA and DNA repair processes.

Main Methods:

• In vitro enzymatic assays to assess HELB helicase activity on naked and RPA-coated DNA substrates.
• Cellular assays to evaluate the recruitment of HELB to sites of DNA damage and its effect on homologous recombination.
• Analysis of the interaction between wild-type HELB and D506G mutant HELB with RPA.

Main Results:

• The D506G substitution in HELB, caused by rs75770066, does not affect its enzymatic activity on naked DNA.
• The D506G substitution significantly reduces the unwinding rate of RPA-coated DNA substrates, indicating impaired interaction with RPA.
• D506G HELB exhibits reduced recruitment to DNA damage sites and leads to increased homologous recombination, suggesting impaired cellular function.

Conclusions:

• The rs75770066 polymorphism impairs HELB function by disrupting its interaction with RPA, leading to reduced recruitment to DNA breaks.
• Altered DNA repair dynamics, including increased homologous recombination and potential effects on meiotic recombination, may influence gamete viability.
• These molecular and cellular changes provide a potential mechanism linking the HELB rs75770066 variant to the observed alterations in the age at natural menopause.