Inter-subunit interactions that coordinate Rad51's activities

Grigorescu, Arabela A.; Vissers, Jmh Jan; Ristić, Dejan; Pigli, Ying Z.; Lynch, Thomas W.; Wyman, Claire; Rice, Phoebe A.

doi:10.1093/nar/gkn973

Cited by 14 publications

(18 citation statements)

References 58 publications

(81 reference statements)

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“…We also note that our results differ from those of a previous study wherein a 63-mer oligonucleotide appeared to stimulate the ATPase activity of H352Y (13). To explore this difference, we repeated the ATPase assays in Figure 4 using a 62-mer random-sequence ssDNA oligo in place of 5400-base φX174 ssDNA.…”

Section: Resultscontrasting

confidence: 99%

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Chen

Villanueva

Rould

et al. 2010

Nucleic Acids Research

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Rad51 protein promotes homologous recombination in eukaryotes. Recombination activities are activated by Rad51 filament assembly on ssDNA. Previous studies of yeast Rad51 showed that His352 occupies an important position at the filament interface, where it could relay signals between subunits and active sites. To investigate, we characterized yeast Rad51 H352A and H352Y mutants, and solved the structure of H352Y. H352A forms catalytically competent but salt-labile complexes on ssDNA. In contrast, H352Y forms salt-resistant complexes on ssDNA, but is defective in nucleotide exchange, RPA displacement and strand exchange with full-length DNA substrates. The 2.5 Å crystal structure of H352Y reveals a right-handed helical filament in a high-pitch (130 Å) conformation with P61 symmetry. The catalytic core and dimer interface regions of H352Y closely resemble those of DNA-bound Escherichia coli RecA protein. The H352Y mutation stabilizes Phe187 from the adjacent subunit in a position that interferes with the γ-phosphate-binding site of the Walker A motif/P-loop, potentially explaining the limited catalysis observed. Comparison of Rad51 H352Y, RecA–DNA and related structures reveals that the presence of bound DNA correlates with the isomerization of a conserved cis peptide near Walker B to the trans configuration, which appears to prime the catalytic glutamate residue for ATP hydrolysis.

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Section: Resultscontrasting

confidence: 99%

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Chen

Villanueva

Rould

et al. 2010

Nucleic Acids Research

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“…The Rad51-H352Y mutant has very low ATPase activity that is essentially limited to a single turnover due to defective ADP/ATP exchange (Chen et al 2010). This mutant fails to catalyze DNA strand exchange reactions using either short oligonucleotide or long M13-derived DNA substrates (Chen et al 2010, Grigorescu et al 2009). In addition, Rad51-H352Y fails to displace RPA from ssDNA in the presence of ATP even though it has higher than wild-type affinity for ssDNA (Chen et al 2010).…”

Section: Filament Dynamics: Factors That Promote Presynaptic Filamentioning

confidence: 99%

Presynaptic filament dynamics in homologous recombination and DNA repair

Liu

Ehmsen

Heyer

et al. 2011

Critical Reviews in Biochemistry and Molecular Biology

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Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity.

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“…This suggests that phosphorylation on S192 might affect Rad51 filament formation. Furthermore, in the Rad51 filament structure, a sulphate ion resides in the P-loop, where the middle phosphate group of ATP would bind (Grigorescu et al, 2009), and a phosphate group covalently attached to S192 would also be able to occupy this position ( Fig 4A). To address the potential impact of S192 on Rad51 multimer formation, we expressed wild-type Rad51, Rad51-S192A and Rad51-S192E, purified them from bacteria and monitored their native oligomer states by gel filtration.…”

Section: Rad51 Ser 192 Affects Rad51 Atpase Activitymentioning

confidence: 99%

Regulation of Rad51 function by phosphorylation

et al. 2011

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Rad51 is a key enzyme involved in DNA double-strand break repair by homologous recombination. Here, we show that in response to DNA damage, budding yeast Rad51 is phosphorylated on Ser-192 in a manner that is primarily mediated by the DNA-damage responsive protein kinase Mec1. We show that mutating Rad51 Ser-192 to Ala or Glu confers hypersensitivity to DNA damage and homologous recombination defects. Furthermore, biochemical analyses indicate that Ser-192 is required for Rad51 ATP hydrolysis and DNA binding activity in vitro, while mutation of Ser-192 does not markedly interfere with Rad51 multimer formation. These data suggest a model in which Mec1-mediated phosphorylation of Rad51 Ser-192 in response to DNA damage controls Rad51 activity and DNA repair by homologous recombination.

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Inter-subunit interactions that coordinate Rad51's activities

Cited by 14 publications

References 58 publications

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant

Presynaptic filament dynamics in homologous recombination and DNA repair

Regulation of Rad51 function by phosphorylation

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