Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.     

The USP1/UAF1 complex promotes double-strand break repair through homologous recombination

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1^−/−, UAF1^−/−/−, and USP1^−/− UAF1^−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.     

DNA double-strand break repair by homologous recombination

The induction of double-strand breaks (DSBs) in DNA by exposure to DNA damaging agents, or as intermediates in normal cellular processes, constitutes a severe threat for the integrity of the genome. If not properly repaired, DSBs may result in chromosomal aberrations, which, in turn, can lead to cell death or to uncontrolled cell growth. To maintain the integrity of the genome, multiple pathways for the repair of DSBs have evolved during evolution: homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). HR has the potential to lead to accurate repair of DSBs, whereas NHEJ and SSA are essentially mutagenic. In yeast, DSBs are primarily repaired via high-fidelity repair of DSBs mediated by HR, whereas in higher eukaryotes, both HR and NHEJ are important. In this review, we focus on the functional conservation of HR from fungi to mammals and on the role of the individual proteins in this process.     

DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSB) are presumed to be the most deleterious DNA lesions as they disrupt both DNA strands. Homologous recombination (HR), single-strand annealing, and non-homologous end-joining are considered to be the pathways for repairing DSB. In this review, we focus on DSB repair by HR. The proteins involved in this process as well as the interactions among them are summarized and characterized. The main emphasis is on eukaryotic cells, particularly the budding yeast Saccharomyces cerevisiae and mammals. Only the RAD52 epistasis group proteins are included.     

The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro

Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.     

DNA double strand break repair in mammalian cells

Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

Mechanistic insights into the activation of Rad51-mediated strand exchange from the structure of a recombination activator, the Swi5-Sfr1 complex

Rad51 forms a helical filament on single-stranded DNA and promotes strand exchange between two homologous DNA molecules during homologous recombination. The Swi5-Sfr1 complex interacts directly with Rad51 and stimulates strand exchange. Here we describe structural and functional aspects of the complex. Swi5 and the C-terminal core domain of Sfr1 form an essential activator complex with a parallel coiled-coil heterodimer joined firmly together via two previously uncharacterized leucine-zipper motifs and a bundle. The resultant coiled coil is sharply kinked, generating an elongated crescent-shaped structure suitable for transient binding within the helical groove of the Rad51 filament. The N-terminal region of Sfr1, meanwhile, has an interface for binding of Rad51. Our data suggest that the snug fit resulting from the complementary geometry of the heterodimer activates the Rad51 filament and that the N-terminal domain of Sfr1 plays a role in the efficient recruitment of the Swi5-Sfr1 complex to the Rad51 filaments.     

SYCE2 is required for synaptonemal complex assembly, double strand break repair, and homologous recombination

Synapsis is the process by which paired chromosome homologues closely associate in meiosis before crossover. In the synaptonemal complex (SC), axial elements of each homologue connect through molecules of SYCP1 to the central element, which contains the proteins SYCE1 and -2. We have derived mice lacking SYCE2 protein, producing males and females in which meiotic chromosomes align and axes form but do not synapse. Sex chromosomes are unaligned, not forming a sex body. Additionally, markers of DNA breakage and repair are retained on the axes, and crossover is impaired, culminating in both males and females failing to produce gametes. We show that SC formation can initiate at sites of SYCE1/SYCP1 localization but that these points of initiation cannot be extended in the absence of SYCE2. SC assembly is thus dependent on SYCP1, SYCE1, and SYCE2. We provide a model to explain this based on protein-protein interactions.     

Involvement of a periplasmic protein kinase in DNA strand break repair and homologous recombination in Escherichia coli

The involvement of signal transduction in the repair of radiation-induced damage to DNA has been known in eukaryotes but remains understudied in bacteria. This article for the first time demonstrates a role for the periplasmic lipoprotein (YfgL) with protein kinase activity transducing a signal for DNA strand break repair in Escherichia coli. Purified YfgL protein showed physical as well as functional interaction with pyrroloquinoline-quinone in solution and the protein kinase activity of YfgL was strongly stimulated in the presence of pyrroloquinoline-quinone. Transgenic E. coli cells producing Deinococcus radiodurans pyrroloquinoline-quinone synthase showed nearly four log cycle improvement in UVC dark survival and 10-fold increases in gamma radiation resistance as compared with untransformed cells. Pyrroloquinoline-quinone enhanced the UV resistance of E. coli through the YfgL protein and required the active recombination repair proteins. The yfgL mutant showed higher sensitivity to UVC, mitomycin C and gamma radiation as compared with wild-type cells and showed a strong impairment in homologous DNA recombination. The mutant expressing an active YfgL in trans recovered the lost phenotypes to nearly wild-type levels. The results strongly suggest that the periplasmic phosphoquinolipoprotein kinase YfgL plays an important role in radiation-induced DNA strand break repair and homologous recombination in E. coli.     

Differential usage of non-homologous end-joining and homologous recombination in double strand break repair

Repair of DNA double strand breaks (DSBs) plays a critical role in the maintenance of the genome. DSB arise frequently as a consequence of replication fork stalling and also due to the attack of exogenous agents. Repair of broken DNA is essential for survival. Two major pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to deal with these lesions, and are conserved from yeast to vertebrates. Despite the conservation of these pathways, their relative contribution to DSB repair varies greatly between these two species. HR plays a dominant role in any DSB repair in yeast, whereas NHEJ significantly contributes to DSB repair in vertebrates. This active NHEJ requires a regulatory mechanism to choose HR or NHEJ in vertebrate cells. In this review, we illustrate how HR and NHEJ are differentially regulated depending on the phase of cell cycle and on the nature of the DSB.     

XRCC1 and DNA strand break repair

DNA single-strand breaks can arise indirectly, as normal intermediates of DNA base excision repair, or directly from damage to deoxyribose. Because single-strand breaks are induced by endogenous reactive molecules such as reactive oxygen species, these lesions pose a continuous threat to genetic integrity. XRCC1 protein plays a major role in facilitating the repair of single-strand breaks in mammalian cells, via an ability to interact with multiple enzymatic components of repair reactions. Here, the protein-protein interactions facilitated by XRCC1, and the repair processes in which these interactions operate, are reviewed. Models for the repair of single-strand breaks during base excision repair and at direct breaks are presented.     

Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

Homologous recombination (HR) represents a major error-free pathway to eliminate pre-carcinogenic chromosomal lesions. The DNA strand invasion reaction in HR is mediated by a helical filament of the Rad51 recombinase assembled on single-stranded DNA that is derived from the nucleolytic processing of the primary lesion. Recent studies have found that the human and mouse Swi5 and Sfr1 proteins form a complex that influences Rad51-mediated HR in cells. Here, we provide biophysical evidence that the mouse Swi5-Sfr1 complex has a 1:1 stoichiometry. Importantly, the Swi5-Sfr1 complex, but neither Swi5 nor Sfr1 alone, physically interacts with Rad51 and stimulates Rad51-mediated homologous DNA pairing. This stimulatory effect stems from the stabilization of the Rad51-ssDNA presynaptic filament. Moreover, we provide evidence that the RSfp (rodent Sfr1 proline rich) motif in Sfr1 serves as a negative regulatory element. These results thus reveal an evolutionarily conserved function in the Swi5-Sfr1 complex and furnish valuable information as to the regulatory role of the RSfp motif that is specific to the mammalian Sfr1 orthologs.     

Aging impairs double‐strand break repair by homologous recombination in Drosophila germ cells

Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double‐strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error‐free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I‐SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR‐white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.     

A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair

XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination.     

DNA strand break repair and neurodegeneration

A number of DNA repair disorders are known to cause neurological problems. These disorders can be broadly characterised into early developmental, mid-to-late developmental or progressive. The exact developmental processes that are affected can influence disease pathology, with symptoms ranging from early embryonic lethality to late-onset ataxia. The category these diseases belong to depends on the frequency of lesions arising in the brain, the role of the defective repair pathway, and the nature of the mutation within the patient. Using observations from patients and transgenic mice, we discuss the importance of double strand break repair during neuroprogenitor proliferation and brain development and the repair of single stranded lesions in neuronal function and maintenance.     

Inhibition of homologous recombination by hyperthermia shunts early double strand break repair to non-homologous end-joining

In S and G2 phase mammalian cells DNA double strand breaks (DSBs) can potentially be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). Results of several studies suggest that these two mechanistically distinct repair pathways can compete for DNA ends. Because HR and NHEJ differ with respect to error susceptibility, generation of chromosome rearrangements, which are potentially carcinogenic products of DSB repair, may depend on the pathway choice. To investigate this hypothesis, the influence of HR and NHEJ inhibition on the frequencies of chromosome aberrations in G2 phase cells was investigated. SW-1573 and RKO cells were treated with mild (41 °C) hyperthermia in order to disable HR and/or NU7441/cisplatin to inactivate NHEJ and frequencies of chromosomal fragments (resulting from unrepaired DSBs) and translocations (products of erroneous DSB rejoining) were studied using premature chromosome condensation (PCC) combined with fluorescence in situ hybridization (FISH).It is shown here that temporary inhibition of HR by hyperthermia results in increased frequency of ionizing-radiation (IR)-induced chromosomal translocations and that this effect is abrogated by NU7441- or cisplatin-mediated inhibition of NHEJ. The results suggest that in the absence of HR, DSB repair is shifted to the error-prone NHEJ pathway resulting in increased frequencies of chromosomal rearrangements. These results might be of consequence for clinical cancer treatment approaches that aim at inhibition of one or more DSB repair pathways.     

Role of Elg1 protein in double strand break repair

The inaccurate repair of DNA double-strand breaks (DSBs) can result in genomic instability, and additionally cell death or the development of cancer. Elg1, which forms an alternative RFC-like complex with RFC2-5, is required for the maintenance of genome stability in Saccharomyces cerevisiae, and its function has been linked to DNA replication or damage checkpoint response. Here, we show that Elg1 is involved in homologous recombination (HR)-mediated DSB repair. Mutants of elg1 were partially defective in HR induced by methylmethanesufonate (MMS) and phleomycin. Deletion of ELG1 resulted in less efficient repair of phleomycin-induced DSBs in G₂/M phase-arrested cells. During HR between MAT and HML loci, Elg1 associated with both the MAT locus near the HO endonuclease-induced DSB site, and the HML homologous donor locus. The association of Elg1 with the MAT locus was not dependent on Rad52. However, Elg1 association with the HML locus depended on Rad52. Importantly, we found that two of the later steps in HR-mediated repair of an HO endonuclease-induced DSB, primer extension after strand invasion and ligation, were less efficient in elg1 mutants. Our results suggest that Elg1 is involved in DSB repair by HR.