A substantial fraction of the eukaryotic genome contains repetitive DNA. Therefore, recombination between tandem repeat sequences likely provides an important opportunity for DNA DSB repair in eukaryotic cells. Furthermore, the same mechanism accounts for targeted gene replacement in yeast and mammals (Langston and Symington, 2005
; Niedernhofer et al., 2001
). Even though ten genes were identified for their contributions to SSA, no prior attempt has been made to systematically reveal genetic components involved in SSA. Here we report a novel genetic screen coupling a plasmid-based SSA assay with a microarray-based technique using yeast nonessential deletion pools. The screen uncovered the role of eight SSA genes, among which one is a new recombination gene: SAW1
. Further analyses of the SAW1
gene products in different steps of SSA revealed that both Slx4 and Saw1 are needed for efficient Rad1/Rad10-dependent 3’-flap cleavage during homologous recombination. Saw1 is also essential in targeting Rad1 to the recombination intermediates in vivo
. The results provide insights into how the Rad1/Rad10 endonuclease recognizes and processes recombination intermediates in a manner distinct from its function in the UV damage repair.
We developed a plasmid-based assay that quantitatively measure the recombination between direct repeat sequences and adapted it to a genome-wide screen that successfully revealed five known SSA genes (RAD1, RAD10, RAD59, MSH2
, a new SSA gene identified most recently (Flott et al., 2007
), and the SAW1
gene previously not linked to SSA. Slx4 catalyzes SSA independently of Slx1, a well-known component for the Slx1-Slx4 endonuclease complex (Flott et al., 2007
). The biochemical study showed that the Slx1-Slx4 complex catalyzes 5’-endonucleolytic cleavage of the replication intermediates, whereas Slx4 alone has a weak 3’-endonuclease activity (Fricke and Brill, 2003
). The Slx1-independent role of Slx4 in the genotoxic damage repair has been proposed based on the hypersensitivity to MMS in the slx4
Δ mutant but not in the slx1
Δ mutant (Roberts et al., 2006
). Slx4 but not Slx1 is post-translationally modified by Rad53 kinase and required for rDNA stability (Coulon et al., 2004
; Flott and Rouse, 2005
; Kaliraman and Brill, 2002
We found that Slx4 is needed for an efficient removal of 3’-flap intermediates during homologous recombination. How does Slx4 stimulate 3’-flap intermediate processing? Slx4 may directly participate in removal of 3’-flaps through its weak 3’ endonuclease activity reported in biochemical study (Fricke and Brill, 2003
). Alternatively, Slx4 may assist in formation or stabilization of 3’-flap intermediates and thus indirectly participate in the 3’-flap cleavage. As the analysis of 3’-flap cleavage in the slx4
Δ, or rad1
Δ strain implicated almost identical substrate specificity for 3’-flap removal with Rad1/Rad10 complex, contribution as a 3’ endonuclease to remove 3’-flap is unlikely. More biochemical studies are needed to elaborate either of these two models further to define the relevant enzymatic activity of Slx4 in homologous recombination.
We identified SAW1
as a new gene responsible for 3’-flap removal that operates in conjunction with Rad1/Rad10 endonuclease. The role of Saw1 in SSA and a subset of gene conversion processes was confirmed by five different recombination assays: the plasmid-based SSA assay, two chromosome-based SSA assays, a plasmid-based gene conversion assay with 3’-flap intermediates, and monitoring of 3’-flap intermediates in vivo
. Saw1 likely performs this role by physically interacting with the Rad1/Rad10 endonuclease complex. Saw1 proteins also interact with Msh2/Msh3 and Rad52, and the interaction with Rad52 is induced by DNA damage. Size fractionation of cell extracts using sucrose density gradient ultracentrifugation suggests that Saw1 proteins form a large molecular weight complex and co-eluted at the same fractions with the Rad1 proteins (Supplemental Fig. 5
). Furthermore, Saw1 is unstable in a rad1
Δ strain, suggesting that all Saw1 likely exists as a complex with Rad1/Rad10 proteins (see Supplemental Fig. 6
). Expression of saw1
mutants that disrupt physical interaction with Rad1, but retain interaction with Msh2 and Rad52 almost completely disabled SSA and gene conversion requiring Rad1/Rad10-dependent 3’-flap removal. Altogether, we propose that Saw1 is a new component of the 3’-flap removal complex comprising Rad1/Rad10, Msh2/Msh3 and Rad52 proteins.
Deletion of SAW1 causes a marked deficiency in the 3’-flap removal step during recombination. Saw1 apparently operates in the same pathway with the Rad1/Rad10 complex and Slx4 proteins because mutants deleting both SAW1 and RAD1 or SAW1 and SLX4 exhibited 3’-flap removal defect indistinguishable from each single gene deletion mutant. Furthermore, analysis of 3’-flap cleavage in the absence of SAW1 suggests identical substrate specificity with the Rad1/Rad10 complex and Slx4 proteins. However, the primary difference still exists between these proteins for their role in the repair of UV-induced DNA lesions: Rad1/Rad10 is essential for NER, but Saw1 is completely dispensable. Additionally, saw1 and slx4 mutations have opposing effects on the frequency of recombination at rDNA, despite their common molecular defect: lack of 3′-flap cleavage. We propose that one of these two proteins, besides its role in 3’-flap processing, has a distinct role in the regulation of recombination at the rDNA.
We showed that the rad1-D825A mutant with an amino acid substitution at the nuclease active site conserved between Rad1 and XPF fails to cleave 3’-flaps and produce SSA product but associates with 3’-flap intermediates in a Saw1-dependent manner in vivo
, whereas wild type Rad1 is undetectable by ChIP assay at the 3’-flap intermediates. The results are highly congruent with the model that Rad1/Rad10 endonuclease only transiently associates with its substrates and that Saw1 is critical for this interaction. Upon successful cleavage of 3’-flaps from annealed intermediates, Rad1 may not sustain interaction with the recombination intermediates because Rad1 can no longer stably associate with the remaining duplex DNA or be removed along with the disjoined flaps due to tight association with ssDNA. Saw1 may target Rad1/Rad10 through physical association with Rad1/Rad10 and other recombination proteins (including RPA, Rad52 and/or Rad59) that bind to ssDNA after 5’ nucleolytic processing of the DNA break. In mammals, RPA and Rad52 are all implicated in modulating 3’-flap removal reaction (de Laat et al., 1998
; Motycka et al., 2004
). Saw1 and its interaction with Rad52 and Rad1 seem well suited for this purpose. Indeed, saw1
mutants that fail to interact with Rad1, but retain interaction with Msh2 and Rad52 are severely deficient in 3’-flap cleavage in vivo.
Alternatively, Saw1 may target Rad1/Rad10 to the recombination intermediate by recognizing and binding directly to DNA substrates bearing distinct structural motif typical of 3’-flap cleavage.
We initially anticipated that our microarray-based genetic screen should reveal SSA factors in different steps of SSA: end resection, strand annealing, 3’-flap cleavage, gap synthesis, and ligation. However, the recombination defect from all identified SSA mutants except rad59
Δ causes accumulation of 3’-flap recombination intermediates. Multiple explanations may account for this unexpected result. First, other recombination reactions prior to the 3’-flap cleavage may be carried out by redundant mechanisms and inactivation of one gene may not produce sufficient defects identifiable in the screen. In line with this premise, end resection depends on both Mre11/Rad50/Xrs2 complex and ExoI protein besides yet unidentified nuclease(s) (Moreau et al., 2001
). Therefore, inactivation of either MRE11
did not reduce SSA to the level lower than those of false positives albeit the repair kinetics are significantly delayed (Ivanov et al., 1994
). Secondly, HR factors involved in other steps of SSA may be essential for cell survival. Because we used nonessential gene deletion pools for the screen, we could not test the role of essential genes in SSA. An example of this category may include Cdc9, a likely ligase involved in ligation step of recombination but it is essential for DNA lagging strand synthesis (Game et al., 1979
) and so mutants are not represented in the pool. Third, slow growth of certain deletion mutants may hamper a reliable assessment of their SSA deficiency by the screen employed here. For instance, the weak hybridization signals from RAD52
gene deletion mutants reflect their slow growth and likely account for the failure to detect a bigger effect on SSA in our screen. The inability to detect the effect of RAD52
deletion on SSA in the current screen leave open the possibility that additional factors important for efficient SSA may yet be undetected from our screen if the corresponding gene deletion mutants grow significantly slower than normal.
For a potentially simple biochemical reaction of the 3’-flap cleavage during recombination require at least 6 proteins among which two (Rad1 and Slx4) have a structure-specific endonuclease activity. Other proteins likely assist in recognition or stabilization of the 3’-flap structure recombination intermediates and position nuclease(s) to the proper target sequences. We propose that analogous biochemical activities are required for equivalent reactions in mammals. Additional molecular details how these proteins and complexes catalyze 3’-flap cleavage require further analysis on the biochemical properties of each SSA component and reconstitution of 3’-flap cleavage reaction using purified proteins and 3’-flap recombination intermediates.