Enemark, E. J. | bioRxiv Science

bioRxiv2024

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

HELB is a human helicase involved in initiation of DNA replication, the replication stress response, and regulation of double-strand DNA break repair. rs75770066 is a rare SNP in the HELB gene that affects age at natural menopause. rs75770066 results in a D506G substitution in an acidic patch within the 1A domain of the helicase that is known to interact with RPA. We found that this amino acid change dramatically impairs the cellular function of HELB. D506G-HELB exhibits impaired interaction with RPA, which likely results in the effects of rs75770066 as this reduces recruitment of HELB to sites of DNA damage. Reduced recruitment of D506G-HELB to double-strand DNA breaks and the concomitant increase in homologous recombination likely alters the levels of meiotic recombination, which affects the viability of gametes. Because menopause occurs when oocyte levels drop below a minimum threshold, altered repair of meiotic double-stranded DNA breaks has the potential to directly affect the age at natural menopause.

biochemistry↗

bioRxiv2022

Enteroviral 2C protein is an RNA-stimulated ATPase and uses a two-step mechanism for binding to RNA and ATP

The enteroviral 2C protein is a therapeutic target, but the absence of a mechanistic framework for this enzyme limits our understanding of inhibitor mechanisms. Here we use poliovirus 2C and a derivative thereof to elucidate the first biochemical mechanism for this enzyme and confirm the applicability of this mechanism to other members of the enterovirus genus. Our biochemical data are consistent with a dimer forming in solution, binding to RNA, which stimulates ATPase activity by increasing the rate of hydrolysis without impacting affinity for ATP substantially. Both RNA and DNA bind to the same or overlapping site on 2C, driven by the phosphodiester backbone, but only RNA stimulates ATP hydrolysis. We propose that RNA binds to 2C driven by the backbone, with reorientation of the ribose hydroxyls occurring in a second step to form the catalytically competent state. 2C also uses a two-step mechanism for binding to ATP. Initial binding is driven by the and {beta} phosphates of ATP. In the second step, the adenine base and other substituents of ATP are used to organize the active site for catalysis. These studies provide the first biochemical description of determinants driving specificity and catalytic efficiency of a picornaviral 2C ATPase.

biochemistry↗