NMR analyses on N-hydroxymethylated nucleobases - implications for formaldehyde toxicity and nucleic acid demethylases

S. Shishodia, D. Zhang, A. H. El-Sagheer, T. Brown, T. D. W. Claridge, C. J. Schofield and R. J. Hopkinson. Org. Biomol. Chem. 16 (21), 4021-4032, 2018.


The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) with similar affinities, raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By fitting detailed polymerisation models informed by our experimental datasets, we show that RAD51 is a mechanosensor that exhibits a larger polymerisation rate constant on flexible ssDNA compared to rigid ssDNA or dsDNA. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in recruiting repair factors that multimerise at sites of DNA damage.