Abstract
Homologous recombination (HR) is essential for maintaining genome stability. Although Rad51 is the key protein that drives HR, multiple auxiliary factors interact with Rad51 to potentiate its activity. Here, we present an interdisciplinary characterization of the interactions between Rad51 and these factors. Through structural analysis, we identified an evolutionarily conserved acidic patch of Rad51. The neutralization of this patch completely abolished recombinational DNA repair due to defects in the recruitment of Rad51 to DNA damage sites. This acidic patch was found to be important for the interaction with Rad55-Rad57 and essential for the interaction with Rad52. Furthermore, biochemical reconstitutions demonstrated that neutralization of this acidic patch also impaired the interaction with Rad54, indicating that a single motif is important for the interaction with multiple auxiliary factors. We propose that this patch is a fundamental motif that facilitates interactions with auxiliary factors and is therefore essential for recombinational DNA repair.
Highlights
Exogenous factors such as ionizing radiation and genotoxic chemicals can cause DNA damage
Mostly negatively charged regions were found on the opposite face of S. pombe Rad51 (SpRad51), including a protruding acidic patch, which we refer to as the PAP hereafter (Figure 1A, right)
To Rad51-EED, we saw a reproducible reduction in the co-IP of Rad51-E206K with Sfr1, but this difference is relatively subtle. These results indicate that PAP mutations do not impair the potentiation of Rad51 by Swi5-Sfr1, leading us to conclude that the severe DNA damage sensitivity of the rad51-EED strain is unlikely to be due to a defect in the interaction with Swi5-Sfr1
Summary
Exogenous factors such as ionizing radiation and genotoxic chemicals can cause DNA damage. Endogenous processes such as DNA replication and cellular metabolism can damage DNA (Lambert and Carr, 2013). A severe form of DNA damage is a DNA double-strand break (DSB), in which a single normal chromosome is separated into two pathological chromosomes. Homologous recombination (HR) is a major mechanism responsible for accurately repairing DSBs. HR is critically important for DNA replication (Ait Saada et al, 2018). Defects in HR lead to genome instability, which drives human diseases such as cancer (Prakash et al, 2015)
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