Sws1 is a conserved regulator of homologous recombination in eukaryotic cells

Abstract

Rad52-dependent homologous recombination (HR) is regulated by the antirecombinase activities of Srs2 and Rqh1/Sgs1 DNA helicases in fission yeast and budding yeast. Functional analysis of Srs2 in Schizosaccharomyces pombe led us to the discovery of Sws1, a novel HR protein with a SWIM-type Zn finger. Inactivation of Sws1 suppresses the genotoxic sensitivity of srs2Delta and rqh1Delta mutants and rescues the inviability of srs2Delta rqh1Delta cells. Sws1 functions at an early step of recombination in a pro-recombinogenic complex with Rlp1 and Rdl1, two RecA-like proteins that are most closely related to the human Rad51 paralogs XRCC2 and RAD51D, respectively. This finding indicates that the XRCC2-RAD51D complex is conserved in lower eukaryotes. A SWS1 homolog exists in human cells. It associates with RAD51D and ablating its expression reduces the number of RAD51 foci. These studies unveil a conserved pathway for the initiation and control of HR in eukaryotic cells.

Figure 1

SWIM domain of Sws1 and MMS sensitivity of sws1Δ mutants. (A) SWIM domain of Sws1. The potential metal-chelating residues defined by Makarova et al (2002) are shown. The predicted α-helix and β-strands are highlighted. The secondary structure prediction was obtained using the Jpred program. (B) DNA damage survival assay. Four-fold serial dilutions of wt (PR109) and sws1Δ (VM3723) cells were plated on YES media and exposed to 0.02% MMS. Photographs were taken after 4 days at 32°C.

Figure 2

Effect of sws1⁺ deletion on survival of srs2Δ and rqh1Δ mutants. (A) Spot assay of wt (PR109), sws1Δ (VM3723), srs2Δ (VM3724) and sws1Δ srs2Δ (VM3721) strains. Four-fold serial dilutions of each strain were plated on YES or YES supplemented with 6 μM camptothecin. Photographs were taken after 4 days at 32°C. (B) DNA damage sensitivity of wt (PR109), sws1Δ (VM3723), rqh1Δ (SC3250), and sws1Δ rqh1Δ (VM3722) cells. Four-fold serial dilutions were plated onto YES plates and treated with the indicated DNA damaging agents. Photographs were taken after 4 days at 32°C. (C) Percentages of dividing cells with ‘cut' phenotype observed in wt (PR109), sws1Δ (VM3723), srs2Δ (VM3724), rqh1Δ (SC3250), sws1Δ srs2Δ (VM3721) and sws1Δ rqh1Δ (VM3722) strains at the indicated time points after release from 5-h-incubation in YES supplemented with 12 mM HU.

Figure 3

Deletion of sws1⁺ rescues the slow growth of srs2Δ rqh1Δ double mutants. (A) Spot assay of wt (PR109), rqh1Δ (SC3250), srs2Δ (VM3724), sws1Δ (VM3723), rqh1Δ srs2Δ (VM3756), sws1Δ rqh1Δ (VM3722) sws1Δ srs2Δ (VM3721) and sws1Δ rqh1Δ srs2Δ (VM3720) strains. Four-fold serial dilutions of each strain were plated on YES. Photographs were taken after 4 days at 32°C. The generation times of wt and some of the mutants used in the spot assay is shown. Cell growth of cultures incubated at 32°C in liquid YES was monitored by measuring optical density at 600 nm. (B) Specificity of the rescue. wt (PR109), sws1Δ (VM3723), sws1Δ rqh1Δ (VM3722), sws1Δ srs2Δ (VM3757) and sws1Δ rqh1Δ srs2Δ (VM3735) were transformed with a pREP41x-Sws1 vector and plated on EMM plates with (for overexpression of Sws1) or without thiamine (repression). When sws1⁺ is overproduced, sws1Δ rqh1Δ srs2Δ (VM3735) cells return to the same rates of slow growth as srs2Δ rqh1Δ (VM3756) cells. Photographs were taken after 5 days at 32°C. (C) Comparison between the subsets of double mutants rescued by sws1Δ and the already characterized recombination genes rad22Δ, rhp51Δ and rhp55Δ. Spores derived from the corresponding crosses were plated on YES and the resulting colonies screened by PCR. Crosses giving rise to triple mutants are shown as ‘+'. Crosses in which it was not possible to recover any triple mutant, or synthetic lethal combinations appeared as ‘−'. ‘+/−' indicates synthetic slow growth.

Figure 4

Spontaneous Rad22-YFP foci formation and recombination levels are reduced in sws1Δ background. (A) Cells containing a genomic copy of Rad22-YFP were grown in YES media at 32°C until mid-log phase and their Rad22-YFP foci were quantified. For each strain more than 300 cells were counted. The average percentage of nuclei containing at least one focus is shown. The strains used in this assay were wt (VM3725), sws1Δ (VM3729), rqh1Δ (VM3726), sws1Δrqh1Δ (VM3730), srs2Δ (VM3727), sws1Δsrs2Δ (VM3728). (B) Comparison of the spontaneous recombination frequencies of wt (PS3), sws1Δ (VM3731), rqh1Δ (VM3732) and sws1Δ rqh1Δ (VM3736). Strains containing an ura4⁺ marker flanked by two ade6⁻ heteroalleles were used in this analysis. Recombination frequencies are mean values from three independent assays.

Figure 5

Sws1 is a conserved protein and the SWIM domain is required for its function. (A) Four-fold serial dilutions of wt (PR109), sws1Δ (VM3723), sws1-C152S (VM3734), rqh1Δ (SC3250), sws1Δ rqh1Δ (VM3722) and sws1-C152S rqh1Δ (VM3733) were plated on YES plates (control), YES plates supplemented with 3 mM HU or 0.02% MMS, or YES plates that afterwards were treated with 300 Gy of IR. Photographs were taken after 4 days at 32°C. (B) Model of the Zn-binding region of the SWIM domain in Sws1. The three cysteine residues and the histidine residue predicted to be involved in Zn chelation are indicated. The cysteine residue (Cys152) mutated in sws1-C152S is underlined. (C) Alignment of Sws1 and its orthologs from H. sapiens, M. musculus, A. thaliana, C. albicans and S. cerevisiae. The conserved CxC(x)_nCxH motif and secondary structure elements (upstream β-strands and the downstream α-helix) are shown.

Figure 6

The S. pombe Rad51 paralogs, Rlp1 and Rdl1, show physical and genetic interactions with Sws1. (A) Schematic representation of the sequences of S. pombe Rdl1 and Rlp1 and their orthologs in H. sapiens (Hs), A. thaliana (At) and S. cerevisiae (Sc). A and B represent the Walker A and Walker B domains, respectively. The regions of highest similarity, centered around the Walker A and B domains are highlighted. (B) Alignment of the RAD51D family members from S. cerevisiae, K. waltii, S. pombe, A. thaliana and H. sapiens. The region of similarity between the different proteins (gray-shadowed region from the corresponding proteins in A) is shown. Conserved residues appear highlighted and the position of the Walker B motif is indicated. (C) Coimmunoprecipitation of Sws1, Rdl1 and Rlp1. Cells simultaneously transformed with the appropriate Rdl1-, Rlp1- and/or Sws1-expressing vectors were used in the assay (pREP41x for 2myc tags and pREP42x for TAP tags). Cell extracts were obtained after 21 h of incubation in the absence of thiamine. ^*Indicates the presence of a nonspecific band. (D) Spot assay of wt (PR109), sws1Δ (VM3723), rlp1Δ (VM3741), rdl1Δ (VM3744) and sws1Δrlp1Δrdl1Δ (VM3755) strains. Four-fold serial dilutions of each strain were plated on YES plates (CONTROL) or YES supplemented with 0.02% MMS. Photographs were taken after 4 days at 32°C. (E) Serial dilutions (fourfold) of the indicated strains were plated in YES plates in the presence of different sources of DNA damage. Photographs were taken after 4 days of incubation at 32°C. Strains used in this assays: wt (PR109), sws1Δ (VM3723), rdl1Δ (VM3744), rlp1Δ (VM3741), rqh1Δ (SC3250), sws1Δ rqh1Δ (VM3722), rlp1Δ rqh1Δ (VM3740), rdl1Δ rqh1Δ (VM3745), sws1Δ rlp1Δ rqh1Δ (VM3742) and sws1Δ rdl1Δ rqh1Δ (VM3746).

Figure 7

Analysis of the spontaneous rad22-YFP foci formation and recombination levels in different mutant backgrounds. (A) Cells expressing Rad22-YFP from its genomic locus were grown in YES media at 32°C until mid-log phase and their Rad22-YFP foci were quantified. For each strain, more than 300 cells were counted. The average percentage of nuclei containing at least one focus is shown (one focus). Average percentages of nuclei containing two or more foci are also indicated (>1 focus). Note that the values of wt, sws1Δ and rqh1Δ are derived from multiple experiments and are the same as those shown in Figure 4. (B) Spontaneous recombination frequencies of wt (PS3), sws1Δ (VM3731), rlp1Δ (VM3749), rdl1Δ (VM3750), rhp57Δ (VM3748), rqh1Δ (VM3732) sws1Δ rqh1Δ (VM3736), rlp1Δ rqh1Δ (VM3753) and rdl1Δ rqh1Δ (VM3750). Strains containing a non-tandem repeat of ade6⁻ heteroalleles flanking a functional ura4⁺ gene were used in this study. Recombination frequencies are mean values from three independent assays, and in each assay four independent colonies were tested.

Figure 8

Conservation of Sws1 function in human cells. (A) Physical interaction between 3HA-SWS1, FLAG-RAD51D and myc-XRCC2. HeLa cells were transiently transfected with 3HA-SWS1 and myc-XRCC2 in the presence or absence of FLAG-RAD51D. At 48 h after transfection, lysates and FLAG-immunoprecipitates were probed for the presence of 3HA-SWS1, FLAG-RAD51D and myc-XRCC2. (B) Depletion of 3HA-SWS1 by RNAi. Four pSuper-SWS1-RNAi vectors were constructed to target different regions of the SWS1 coding sequence. Two of these vectors (containing SWS1RNAi3 and SWS1RNAi4) successfully suppressed expression of a cotransfected 3HA-SWS1 construct. Western analyses were performed using antiHA antibodies in cell lysates from cells co-transfected with 3HA-SWS1 and pSUPER vectors containing SWS1RNAi3 or SWS1RNAi4. SWS1 protein levels were substantially reduced when RNAi against SWS1 was used, while no loss of SWS1 was seen when cells were transfected with a control vector (pSuper). Tubulin was used as a loading control. (C, D) Representative Rad51 foci in cells transfected with control (pSuper-HA, which expressed HA siRNA) or pSuper-SWS1RNAi3 and 4. Untreated cells (0 Gy) and cells exposed to gamma radiation (10 Gy) were fixed 5 h after treatment for immunocytochemical analysis of Rad51. Rad51 foci were visualized by immunofluorescence staining with Abcam 13E4 anti-Rad51 monoclonal antibodies (C, left). DAPI counterstaining for cell nuclei is also shown (C, right). Quantification of Rad51 foci (D) shows a decrease in the number of cells containing at least two Rad51 foci both before and after gamma radiation. This reduction in spontaneous and gamma-induced Rad51 foci is associated with reduced SWS1 expression, as it was observed after transfection with two different constructs that cause reduced 3HA-SWS1 expression but not after transfection with the control RNAi vector (pSUPER-HA), which does not affect the number of HeLa cells showing RAD51 foci. 350 cells were counted for each sample. (E) Model for the presynaptic events in HR. Our results support a model in which S. pombe Sws1 acts together with Rlp1 and Rdl1 to promote Rad22 recruitment to the sites of DNA damage. The humans SWS1–RAD51D–XRCC2 is proposed to perform a similar function in recruiting BRCA2. We suggest that the Shu2 group of proteins performs an analogous function in S. cerevisiae. Psy3 is proposed to be the homolog of Rdl1 and RAD51D. Shu1 may be a very distantly related homolog of Rlp1 and XRCC2. Human and fission yeast proteins related to Csm2 have not yet been found. Other proteins known to be involved in HR are also shown. See text for further details. A color version of this figure is available at The EMBO Journal Online.