A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

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.

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by Mec1 and its own autophosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 activation has been well documented, less is known about the processes that counteract its activity along the repair of a DNA adduct. Here, we describe that PP4 phosphatase is required to avoid Rad53 hyper-phosphorylation during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects resection and repair by single-strand annealing, defects that are bypassed by reducing Rad53 hyperphosphorylation. These results confirm that Rad53 phosphorylation is controlled by PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection for their success.

PP4 phosphatase cooperates in recombinational DNA repair by enhancing double-strand break end resection

The role of Rad53 in response to a DNA lesion is central for the accurate orchestration of the DNA damage response. Rad53 activation relies on its phosphorylation by the Mec1 kinase and its own auto-phosphorylation in a manner dependent on the adaptor Rad9. While the mechanism behind Rad53 phosphorylation and activation has been well documented, less is known about the processes that counteract its kinase activity during the response to DNA damage. Here, we describe that PP4 phosphatase dephosphorylates Rad53 during the repair of a double-strand break, a process that impacts on the phosphorylation status of multiple factors involved in the DNA damage response. PP4-dependent Rad53 dephosphorylation stimulates DNA end resection by relieving the negative effect that Rad9 exerts over the Sgs1/Dna2 exonuclease complex. Consequently, elimination of PP4 activity affects DNA resection and repair by single-strand annealing, defects that are bypassed by reducing the hyper-phosphorylation state of Rad53 observed in the absence of the phosphatase. These results confirm that Rad53 is one of the main targets of PP4 during the repair of a DNA lesion and demonstrate that the attenuation of its kinase activity during the initial steps of the repair process is essential to efficiently enhance recombinational DNA repair pathways that depend on long-range resection.

Between sisters: Watching replication-associated recombinational DNA repair

Amarh et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201803020) visualize for the first time the repair of double-strand breaks during DNA replication. As viewed by live-cell fluorescent imaging of Escherichia coli, repair of replication-dependent breaks is extraordinarily rapid and localized within the cell.