Tyrosine phosphorylation stimulates activity of human RAD51 recombinase through altered nucleoprotein filament dynamics

Abstract

The DNA strand exchange protein RAD51 facilitates the central step in homologous recombination, a process fundamentally important for accurate repair of damaged chromosomes, restart of collapsed replication forks, and telomere maintenance. The active form of RAD51 is a nucleoprotein filament that assembles on single-stranded DNA (ssDNA) at the sites of DNA damage. The c-Abl tyrosine kinase and its oncogenic counterpart BCR-ABL fusion kinase phosphorylate human RAD51 on tyrosine residues 54 and 315. We combined biochemical reconstitutions of the DNA strand exchange reactions with total internal reflection fluorescence microscopy to determine how the two phosphorylation events affect the biochemical activities of human RAD51 and properties of the RAD51 nucleoprotein filament. By mimicking RAD51 tyrosine phosphorylation with a nonnatural amino acid, p-carboxymethyl-L-phenylalanine (pCMF), we demonstrated that Y54 phosphorylation enhances the RAD51 recombinase activity by at least two different mechanisms, modifies the RAD51 nucleoprotein filament formation, and allows RAD51 to compete efficiently with ssDNA binding protein RPA. In contrast, Y315 phosphorylation has little effect on the RAD51 activities. Based on our work and previous cellular studies, we propose a mechanism underlying RAD51 activation by c-Abl/BCR-ABL kinases.RAD51 recombinase | c-Abl tyrosine kinase | homologous recombination | single-molecule total internal reflection fluorescence microscopy | phosphorylation T he DNA in the human genome is constantly subjected to damage. This damage is a byproduct of normal cellular metabolism, exposure to radiation, and environmental mutagens (1). Homologous recombination (HR) and the pathways that use the machinery of HR are responsible for the accurate repair of the most deleterious DNA lesions, including double-stranded DNA breaks (DSBs), interstrand DNA cross-links, and damaged replication forks, and thereby contribute to maintenance of the stable genome (2-5). HR also plays an important role in telomere maintenance (6). HR, a process highly conserved throughout evolution, is carried out through a precisely coordinated and tightly regulated series of events. The key step in HR is the assembly of a RecA-family recombinase (phage UvsX, bacterial RecA, archaeal RadA, or eukaryotic RAD51) onto resected single-stranded DNA (ssDNA). The recombinase forms a nucleoprotein filament that then invades homologous duplex DNA, resulting in a displacement loop structure that can be used as a primer for synthesis using the intact duplex as a template. Similar to its bacterial and yeast homologs, the human RAD51 binds ssDNA in an ATP-dependent manner (7). The nucleoprotein filament formed by the ATP-bound RAD51 is arranged such that each RAD51 monomer binds three nucleotides, forming the pairing unit in these reactions (8). Beyond these basic attributes, the characteristics of human RAD51 protein differ significantly from its archaeal, bacterial, and yeast homologs.The critical role of HR requires all steps of this process ...

Observation and Analysis of RAD51 Nucleation Dynamics at Single-Monomer Resolution

“…A flow experiment, like the one described in this chapter, follows the very first steps in the RAD51 nucleoprotein filament assembly, namely, a formation of a stable nucleus from which the filament can then grow. In the case of human RAD51 under conditions permitting ATP hydrolysis, filament nucleation occurs through a dynamic association and dissociation of RAD51 dimers (Subramanyam et al, 2016). Similar approaches have been applied to other RecA-family recombinases (including yeast Rad51 protein (Qiu et al, 2013), and RecA (Joo et al, 2006)), albeit with different kinetics and via different oligomeric species.…”

“…In the analysis of RAD51 nucleation dynamics, the lower bounds plateaued starting with the HMM that was composed of four states, so we therefore used this four-state HMM in the subsequent analysis. With four distinct conformational states of the nucleating filament, this suggests that the nucleating species of RAD51 is a dimer (Subramanyam et al, 2016). …”

“…Binding and dissociation of the recombinase results in increases and decreases to the length of the DNA, respectively, which in turn causes decreases and increases in the FRET efficiency ( E FRET ) signal (a measure of non-radiative energy transfer from the FRET donor to the FRET acceptor), respectively. This approach has been applied to study RecA (Joo et al, 2006), yeast Rad51 (Qiu et al, 2013), and human RAD51 (Subramanyam et al, 2016) nucleation on ssDNA. These studies showed that while nucleation by RecA and yeast Rad51 occurs mainly via addition of RecA (Joo et al, 2006) or Rad51 (Qiu et al, 2013) monomers, human RAD51 nucleates on the ssDNA primarily via addition of RAD51 dimers (Subramanyam et al, 2016).…”

“…This approach has been applied to study RecA (Joo et al, 2006), yeast Rad51 (Qiu et al, 2013), and human RAD51 (Subramanyam et al, 2016) nucleation on ssDNA. These studies showed that while nucleation by RecA and yeast Rad51 occurs mainly via addition of RecA (Joo et al, 2006) or Rad51 (Qiu et al, 2013) monomers, human RAD51 nucleates on the ssDNA primarily via addition of RAD51 dimers (Subramanyam et al, 2016). In this chapter, we provide a protocol for the observation, analysis, and interpretation of human RAD51 filament formation by single-molecule FRET.…”

Evaluation of the Kinetic Property of Single-Molecule Junctions by Tunneling Current Measurements

“…For example, through an HMM analysis of the fluorescence resonance energy transfer, the probability of a conformational change of a single biomolecule was determined. 12,13 In nanopore experiments, an HMM analysis revealed the probability of passing of the target molecule through the nanopore.…”