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. 2020 Aug 5;10(8):2811-2818.
doi: 10.1534/g3.120.401324.

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes of Saccharomyces cerevisiae Bloom's Helicase Homolog Sgs1 SUMOylation-Associated Mutants

Matan Cohen  1   2 , Michael Lichten  3
Affiliations

C-Terminal HA Tags Compromise Function and Exacerbate Phenotypes of Saccharomyces cerevisiae Bloom's Helicase Homolog Sgs1 SUMOylation-Associated Mutants

Matan Cohen et al. G3 (Bethesda). .

Abstract

The Sgs1 helicase and Top3-Rmi1 decatenase form a complex that affects homologous recombination outcomes during the mitotic cell cycle and during meiosis. Previous studies have reported that Sgs1-Top3-Rmi1 function is regulated by SUMOylation that is catalyzed by the Smc5-Smc6-Mms21 complex. These studies used strains in which SGS1 was C-terminally tagged with three or six copies of a human influenza hemagglutinin-derived epitope tag (3HA and 6HA). They identified SGS1 mutants that affect its SUMOylation, which we will refer to as SGS1 SUMO-site mutants. In previous work, these mutants showed phenotypes consistent with substantial loss of Sgs1-Top3-Rmi1 function during the mitotic cell cycle. We find that the reported phenotypes are largely due to the presence of the HA epitope tags. Untagged SGS1 SUMO-site mutants show either wild-type or weak hypomorphic phenotypes, depending on the assay. These phenotypes are exacerbated by both 6HA and 3HA epitope tags in two different S. cerevisiae strain backgrounds. Importantly, a C-terminal 6HA tag confers strong hypomorphic or null phenotypes on an otherwise wild-type Sgs1 protein. Taken together, these results suggest that the HA epitope tags used in previous studies seriously compromise Sgs1 function. Furthermore, they raise the possibilities either that sufficient SUMOylation of the Sgs1-Top3-Rmi1 complex might still occur in the SUMO-site mutants isolated, or that Smc5-Smc6-Mms21-mediated SUMOylation plays a minor role in the regulation of Sgs1-Top3-Rmi1 during recombination.

Keywords: HA protein tags; Intramural Research Program of the NIH; Key phrases: Sgs1; SUMO; Smc5-Smc6-Mms21; homologous recombination; meiosis.

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Figures

Figure 1
Figure 1
SGS1 SUMO-site mutants and allelic replacement strategy. A) Map of the Sgs1 protein and the SUMO-site mutants used in this study. Top–K to R mutations used in Bonner et al. (2016) and Bermúdez-López et al. (2016); mutation in blue is common to both studies. sgs1-6KR is called sgs1-3KR in Bermúdez-López et al. (2016). Bottom—SUMO-interaction module (SIM) mutants. Residues in red were replaced by alanine in the indicated report. Sgs1 domains are: AR1, AR2—acidic regions 1 and 2; RQC—RecQ C-terminal domain; HRDC—helicase-and-RNAseD-like-C-terminal domain. Numbers indicate amino acid residues. B) Allelic replacement strategy used to insert SGS1 SUMO-site mutants in the genome of SK1 strains. Plasmids containing mutants were digested to release the indicated fragment, which used homologous sequences up- and downstream of the SGS1 ORF to direct integration. Numbers indicate nucleotides relative to the SGS1 ORF start (negative numbers) and the SGS1 ORF end (positive numbers). Promoters and terminators are indicated by the prefixes “p” and “t,” respectively. Construct segments are not drawn to scale. C) Strategy used to remove the 3xHA tag from SGS1 mutants in W303 strains from Bonner et al. (2016). The indicated PCR fragment, which contains SGS1 sequences 3′ of mutant-containing sequences, was used to replace sequences downstream of SGS1. Numbers are as in (B). Construct segments are not drawn to scale.
Figure 2
Figure 2
C-terminal HA tags exacerbate the mitotic phenotypes of SGS1 SUMO-site mutants. In all panels, “SGS1” indicates an allelic replacement with wild-type sequences in the construct shown in Figure 1; “Wild type” indicates the unmodified endogenous SGS1 locus. A) Genetic interactions between slx4∆ and SGS1 SUMO-site mutants in SK1 strains. Two representative tetrads from the indicated heterozygous diploid are shown after grown for 2 days at 30°C. B) As in A, except with W303 strains from Bonner et al. (2016). C) MMS and HU sensitivity, in SK1, of SGS1 SUMO-site mutants used by Bermúdez-López et al. (2016), with and without 6HA tags. 10-fold dilution series were spotted and allowed to grow 2-3 days at 30°C. D) As in C, but with SUMO-site mutants used by Bonner et al. (2016) without an epitope tag. E) As in D, but with tagged and untagged SUMO-site mutants in W303.
Figure 3
Figure 3
SGS1 SUMO-site mutants retain STR meiotic function. In all panels, “wild type” denotes an unmodified SGS1 locus; “SGS1” denotes the allelic replacement construct (see Figure 1) containing wild-type SGS1 sequences. A) Spore viability of diploids homozygous for the indicated SGS1 allele. Error bars represent the 95% confidence interval. **** indicates P < 0.0001. B) Spore viability of diploids homozygous for msh4∆ and the indicated SGS1 allele; other details as in A). C) Meiotic progression in diploids homozygous for the indicated genotype. The number of nuclei per cell was counted; cells with ≥ 2 nuclei were considered to have undergone at least one nuclear division. At least 200 cells were counted per time point in two independent experiments; error bars represent range.
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