Distinct RPA domains promote recruitment and the helicase-nuclease activities of Dna2

Abstract

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

Distinct RPA functions promote eukaryotic DNA replication initiation and elongation

“…To test whether origin unwinding requires the endogenous, multimeric context of RPA, we generated an RPA mutant, RPA ΔOB-FΔWH , that contains all four DNA-binding domains but lacks the protein-interaction domains, Rfa1-OB-F and Rfa2-WH (Figure 4A-B). These domains interact with DNA replication and repair proteins but are not involved in DNA binding (Acharya et al, 2021; Collins and Kelly, 1991; Dornreiter et al, 1992; Han et al, 1999; Mer et al, 2000; Prakash and Borgstahl, 2012; Shen et al, 2022; Wold et al, 1989; Zou and Elledge, 2003). Indeed, we found this mutant bound DNA with near wild-type affinity (Figure 4C).…”

“…Finally, RPA could also stimulate CMG processivity on ssDNA. Although RPA modulates the activity of other helicases through direct interactions (Acharya et al, 2021; Shorrocks et al, 2021), the ability of EcSSB and RPA ΔOB-FΔWH to substitute for RPA suggests a direct protein-protein interaction between RPA and the helicase are not required. Instead, we consider that RPA stimulates CMG by interacting with the excluded strand.…”

Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection

“…We next decided to address the role of RPA itself, as it has served as a respectable indicator of Scc2 recruitment. Integral to the preservation of ssDNA at broken DNA ends, it has been shown that RPA enforces the correct polarity of DNA end resection and promotes the recruitment and activity of Dna2 (65). To this end, we made use of the rfa1-G77E allele, characterized as the Saccharomyces cerevisiae equivalent to the fission yeast rfa1-G78E allele (66,67), shown to exhibit markedly reduced affinity of RPA for short ssDNA fragments, and a slightly reduced affinity for longer stretches.…”

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB

“…For DNA binding experiments with linear single-stranded DNA, oligonucleotide X12-3 HJ3 (93 nt) was labeled at the 5' terminus with [γ-32P] (Hartmann-Analytic) and T4 polynucleotide kinase (New England Biolabs), according to standard protocols 65 A major portion of the DNA unwinding experiments were performed using 5' overhang-M13 based circular DNA. This was prepared using oligonucleotide (M13-5'-dT 40 overhang) containing a 37 nt-long region complementary to the M13mp18(+) strand (nucleotides 6289-6326), as well as a 40 nt-long tail at the 5' end, which was annealed to M13mp18 ssDNA 39 .…”