Homologous recombination (HR) is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage, including DNA double-strand breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks, enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and elevated cancer predisposition, demonstrating a clear cellular need for recombination. However, recombination can also lead to genome rearrangements. Unrestrained recombination causes undesired endpoints (translocation, deletion, inversion) and the accumulation of toxic recombination intermediates. Evidently, HR must be carefully regulated to match specific cellular needs. Here, we review the factors and mechanistic stages of recombination that are subject to regulation and suggest that recombination achieves flexibility and robustness by proceeding through metastable, reversible intermediates.

BRCA2 is a tumor suppressor that interacts with RAD51 and functions in homologous recombination, but understanding its precise functions has been hampered by difficulties in purifying such a large protein. Now purified full-length human BRCA2 is shown to bind ∼6 molecules of RAD51 and to promote RAD51 binding to RPA-covered ssDNA in a manner stimulated by DSS1. BRCA2 is a tumor suppressor that functions in homologous recombination, a key genomic integrity pathway. BRCA2 interacts with RAD51, the central protein of recombination, which forms filaments on single-stranded DNA (ssDNA) to perform homology search and DNA strand invasion. We report the purification of full-length human BRCA2 and show that it binds to ∼6 RAD51 molecules and promotes RAD51 binding to ssDNA coated by replication protein A (RPA), in a manner that is stimulated by DSS1.

DNA strand exchange, a process that helps to maintain the integrity of the genome, is promoted in eukaryotes by the Rad51 protein. Eukaryotes also have homologues of Rad51 with functions that are less well defined. Wolf-Dietrich Heyer and colleagues show that in yeast, two of these proteins, Rad55 and Rad57, form a complex that promotes recombination by stabilizing the Rad51 filament against the anti-recombinogenic activity of a helicase, Srs2. These results suggest that the Rad51 filament is a reversible intermediate, the stability of which reflects competing actions of other proteins. This mechanism may have relevance for the human RAD51 paralogues that have important roles in tumour suppression and disease. Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion1,2. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined3. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55–Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55–Rad57 heterodimer associates with the Rad51–single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51–Rad55–Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55–Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55–Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops.

Background Acoustic stimulation offers a potential treatment approach for tinnitus but also in-sights in its basic mechanisms by short-term tinnitus suppression called residual inhibition (RI). The effects of RI were found to be depending on intensity, length or sound types covering the individual tinnitus characteristics. In patients with tonal tinnitus RI was increased with amplitude modulated (AM) pure tones at the individual tinnitus frequency while the effects of modulated noise sounds have not been systematically researched. Objectives The aim of the present study was to investigate whether in patients with noise-like tinnitus RI can be increased by AM noise-like stimuli according to the individual tinnitus frequency range. Methods For this purpose the individual tinnitus characteristics (noise-like and tonal tinnitus) were assessed via customizable noise-band matching, in order to generate bandpass filtered stimuli according to the individual tinnitus sound (individualized bandpass filtered sounds; IBP). Subsequent, various stimuli differing in bandpass filtering and AM were tested with respect to their potential to induce RI. Patients were acoustically stimulated with seven different types of stimuli for three minutes each and had to rate the loudness of their tinnitus after each stimuli. Results Results indicate a general efficacy of noise stimuli for the temporary suppression of tinnitus, but no significant differences between AM and unmodulated IBP. Significantly better effects were observed for the subgroup with noise-like tinnitus (n=14), especially directly after stimulation offset. Conclusions The study at hand provides further insights in potential mechanisms behind RI for different types of tinnitus. Beyond that, derived principles may qualify for new or extend current tinnitus sound therapies.

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 (ssDNA). 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 reviewed 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 examined 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 examined the biochemical properties of recombination proteins from four model systems (T4 phage, Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We proposed that the presynaptic filament has evolved to rely on multiple external factors for increased multilevel regulation of HR processes in genomes with greater structural and sequence complexity.