Overlapping roles for Yen1 and Mus81 in cellular Holliday junction processing

Abstract

Eukaryotic Holliday junction (HJ) resolvases have attracted much attention recently with the identification of at least three distinct proteins that can cleave model HJs in vitro. However, the specific DNA structure(s) that these proteins act upon in the cell is unknown. Here, we describe a system in budding yeast to directly and quantitatively monitor in vivo HJ resolution. We found that Yen1 acts redundantly with Mus81, but not Slx1, to resolve a model HJ in vivo. This functional overlap specifically extends to the repair/bypass of lesions that impede the progression of replication forks but not to the repair of double-strand breaks induced by ionizing radiation. Together, these results suggest a direct role for Yen1 in the response to DNA damage and implicate overlapping HJ resolution functions of Yen1 with Mus81 during replication fork repair.

FIGURE 1.

System to directly analyze HJ resolution in vivo. A, depiction of the system used to analyze HJ resolution in vivo. Resolution of the HJ in JM-HJ into an R1-R2 dimer is detected as methionine and histidine prototrophs that arise following JM-HJ transformation into S. cerevisiae. See “Results and Discussion” for details. B, left panel, Southern blot analysis of purified JM, R1, and JM-HJ digested with various enzymes; right panel, representations of JM-HJ molecules present in lanes 7–9. The HJ in JM-HJ is shown in red. C, Southern blot analysis of purified JM-HJ digested with RusA. The asterisk indicates the linear R1-R2 dimer.

FIGURE 2.

R1-R2 dimers can be detected as products of JM-HJ resolution. Left panel, schematic of the predicted R1-R2 dimer with the positions of relevant restriction sites; right panel, Southern analysis of purified JM and genomic DNA from four independent MET17 HIS3 clones arising from JM-HJ transformation digested with various enzymes as indicated. The asterisk indicates monomeric R1 that most likely arose through a post-JM-HJ resolution event through cer-mediated intramolecular recombination of the R1-R2 dimer product.

FIGURE 3.

Yen1 and Mus81 act redundantly to resolve JM-HJ in vivo. A–D, resolution efficiencies of JM-HJ in various strain backgrounds as indicated. Error bars are means ± S.D. from at least three independent experiments.

FIGURE 4.

Yen1 and Mus81 are required for replication fork repair. A–C, DNA damage sensitivity assays of various strains as indicated. Gy, grays; HU, hydroxyurea; CPT, camptothecin; MMS, methyl methanesulfonate.