The possible function of Flp1 in homologous recombination repair in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae Mus81 is a structure-selective endonuclease which constitutes an alternative pathway in parallel with the helicase-topoisomerase Sgs1-Top3-Rmi1 complex to resolve a number of DNA intermediates during DNA replication, repair, and homologous recombination. Previously, it was showed that the N-terminal region of Mus81 was required for its in vivo function in a redundant manner with Sgs1; mus81_Δ120N mutant that lacks the first 120 amino acid residues at the N-terminus exhibited synthetic lethality in combination with the loss of SGS1. In this study, the physiologically important role of the N-terminal region of Mus81 in processing toxic intermediates was further investigated. We examined the cellular defect of sgs1Δmus81_Δ100N cells and observed that although viable, the cells became very sensitive to DNA damaging agents. A single-copy suppressor screening to seek for a factor(s) that could rescue the drug sensitivity of sgs1Δmus81_Δ100N cells was performed and revealed that Flp1, a site-specific recombinase 1 encoded on the 2-micron plasmid was a suppressor. Moreover, Flp1 overexpression could partially suppress the drug sensitivity of mus81Δ cells at 37 °C. Our findings suggest a possible function of Flp1 in coordination with Mus81 and Sgs1 to jointly resolve the branched-DNA structures generated in cells attempting to repair DNA damages.

Keywords: Flp1; Mus81; Sgs1; genetic screening; homologous recombination repair.

Figure 1.. The cellular defect of mus81_Δ80N and mus81_Δ100N. (A) The complementation of sgs1Δmus81Δ synthetic lethality by mus81 mutant alleles. (B) The MMS sensitivity of mus81_Δ80N and mus81_Δ100N. (C) The MMS sensitivity of sgs1Δmus81_Δ80N and sgs1Δmus81_Δ100N. The cells that grew in the presence of 5-FOA (in panel A) were spotted onto without or with indicated amount of MMS or HU concentration.

Figure 2.. Scheme of the single-copy suppressor screening of drug sensitivity of the sgs1Δmus81_Δ100N cells. (1) transformation with yeast genomic library, (2) replica onto HU plates, (3) and (4) drop dilution assay, (5) sequencing. Abbreviations: P, positive control; N, negative control; C1–4, representative colonies number 1 to 4.

Figure 3.. A drop dilution assay examining the transformant colonies that survive in the presence of HU. The sgs1Δmus81_Δ100N cells containing yeast genomic DNA fragment which survived on HU plates after replica step were selected and serial-diluted spotted onto plates without or with 20 mM HU.

Figure 4.. A drop dilution assay to confirm the suppression ability of the candidates. The sgs1Δmus81_Δ100N cells were transformed with extracting plasmids from selected candidates and then serial-diluted spotted onto plates without or with 20 mM HU.

Figure 5.. Transformation of plasmid number 31 can partially suppress the drug sensitivity of mus81Δ at 37 °C. NJY1777 strain was transformed with an empty vector pRS315 or vectors containing MUS81 or screened plasmid number 31. To create sgs1Δ mutant cells, NJY1777 strain was transformed with pRS314 vector containing MUS81 and transformants were grown in the presence of 5-FOA to remove pJM500-URA3-SGS1 plasmid.

Figure 6.. Suppression of drug sensitivity by Flp1 overexpression is specific for mus81 mutants. (A) Overexpression of Flp1 could partially rescue the HU-sensitivity of the sgs1Δmus81_Δ100N cells. FLP1 coding sequence was cloned into pRS315 plasmid and its expression was driven by ADH1 promoter. Empty vector pRS315 or vector containing MUS81 or FLP1 was then transformed into the sgs1Δmus81_Δ100N cells. (B) Overexpression of Flp1 could not suppress the HU-sensitivity of rad52Δ and rad52Δsgs1Δmus81_Δ100N cells. (C) Overexpression of Flp1 could not rescue the HU-sensitivity of wild-type cells.